Methods and compositions for whole genome amplification and genotyping

ABSTRACT

This invention provides methods of amplifying genomic DNA to obtain an amplified representative population of genome fragments. Methods are further provided for obtaining amplified genomic DNA representations of a desired complexity. The invention further provides methods for simultaneously detecting large numbers of typable loci for an amplified representative population of genome fragments. Accordingly the methods can be used to genotype individuals on a genome-wide scale.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Ser. No. 15/816,979, filed Nov. 17,2017, which is a continuation of U.S. application Ser. No. 14/715,440,filed May 18, 2015, which is a continuation of U.S. application Ser. No.10/871,513, filed Jun. 17, 2004, now U.S. Pat. No. 9,045,796, which is acontinuation-in-part of U.S. application Ser. No. 10/681,800, filed onOct. 8, 2003, now abandoned, which is a continuation of U.S. applicationSer. No. 10/600,634, filed on Jun. 20, 2003, now abandoned, the entirecontents of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to genetic analysis and morespecifically to amplification of whole genomes and genotyping based onpluralities of genetic markers spanning genomes.

BACKGROUND OF THE INVENTION

Most of any one person's DNA, some 99.9 percent, is exactly the same asany other person's DNA. The roughly 0.1% difference in the genomesequence accounts for a wide variety of the differences among people,such as eye color and blood group. Genetic variation also plays a rolein whether a person is at risk for getting particular diseases orwhether a person is likely to have a favorable or adverse response to aparticular drug. Single gene differences in individuals have beenassociated with elevated risk for acquiring a variety of diseases, suchas cystic fibrosis and sickle cell disease. More complexinterrelationships among multiple genes and the environment areresponsible for many traits like risk for some common diseases, such asdiabetes, cancer, stroke, Alzheimer's disease, Parkinson's disease,depression, alcoholism, heart disease, arthritis and asthma.

Genetic-based diagnostic tests are available for several highlypenetrant diseases caused by single genes, such as cystic fibrosis. Suchtests can be performed by probing for particular mutations orpolymorphisms in the respective genes. Accordingly, risk for contractinga particular disease can be determined well before symptoms appear and,if desired, preventative measures can be taken. However, it is believedthat the majority of diseases, including many common diseases such asdiabetes, heart disease, cancers, and psychiatric disorders, areaffected by multiple genes as well as environmental conditions. Thus,diagnosis of such diseases based on genetics is considerably morecomplex as the number of genes to be interrogated increases.

Recently, through a variety of genotyping efforts, a large number ofpolymorphic DNA markers have been identified, many of which are believedto be associated with the probability of developing particular traitssuch as risk of acquiring known diseases. Exemplary polymorphic DNAmarkers that are available include single nucleotide polymorphisms(SNPs) which occur at an average frequency of more than 1 per kilobasein human genomic DNA. Many of these SNPs are likely to betherapeutically relevant genetic variants and/or involved in geneticpredisposition to disease. However, current methods for genome-wideinterrogation of SNPs and other markers are inefficient, therebyrendering the identification of useful diagnostic marker setsimpractical.

The ability to simultaneously genotype large numbers of SNP markersacross a DNA sample is becoming increasingly important for geneticlinkage and association studies. A major limitation to whole genomeassociation studies is the lack of a technology to performhighly-multiplexed SNP genotyping. The generation of the completehaplotype map of the human genome across major ethnic groups willprovide the SNP content for whole genome association studies (estimatedat about 200,000-300,000 SNPs). However, currently available genotypingmethods are cumbersome and inefficient for scoring the large numbers ofSNPs needed to generate a haplotype map.

Thus there is a need in the art for methods of simultaneouslyinterrogating large numbers of gene loci on a whole genome scale. Suchbenefits will affect the genomic discovery process and the geneticanalysis of diseases, as well as the genetic analysis of individuals.This invention satisfies this need and provides other advantages aswell. This invention describes and demonstrates a method to performlarge scale multiplexing reactions enabling a new era in genomics.

SUMMARY OF THE INVENTION

In one aspect, the present invention features a method of detecting oneor several typable loci contained within a given genome, where themethod includes the steps of providing an amplified representativepopulation of genome fragments having such typable loci, contacting thegenome fragments with a plurality of nucleic acid probes havingsequences corresponding to the typable loci under conditions whereinprobe-fragment hybrids are formed; and detecting typable loci of theprobe-fragment hybrids. In particular embodiments these nucleic acidprobes are at most 125 nucleotides in length. However, probes having anyof a variety of lengths or sequences can be used as set forth in moredetail below.

In another aspect, the present invention features a method of detectingtypable loci of a genome including the steps of providing an amplifiedrepresentative population of genome fragments that has such typableloci, contacting the genome fragments with a plurality of nucleic acidprobes having sequences corresponding to the typable loci underconditions wherein probe-fragment hybrids are formed; and directlydetecting typable loci of the probe-fragment hybrids.

In a further aspect, the present invention features a method ofdetecting typable loci of a genome including the steps of providing anamplified representative population of genome fragments having thetypable loci; contacting the genome fragments with a plurality ofimmobilized nucleic acid probes having sequences corresponding to thetypable loci under conditions wherein immobilized probe-fragment hybridsare formed; modifying the immobilized probe-fragment hybrids; anddetecting a probe or fragment that has been modified, thereby detectingthe typable loci of the genome.

The invention also provides a method, including the steps of (a)providing a plurality of genome fragments, wherein the plurality ofgenome fragments has at least 100 ug of DNA having a complexity of atleast 1 Gigabases; (b) contacting the plurality of genome fragments witha plurality of different immobilized nucleic acid probes, wherein atleast 500 of the different nucleic acid probes hybridize with genomefragments to form probe-fragment hybrids; and (c) detecting typable lociof the probe-fragment hybrids.

A method of the invention can also include the steps of (a) providing aplurality of genome fragments, wherein the plurality of genome fragmentshas a concentration of at least 1 ug/ul of DNA having a complexity of atleast 1 Gigabases; (b) contacting the plurality of genome fragments witha plurality of different immobilized nucleic acid probes, wherein atleast 500 of the different nucleic acid probes hybridize with genomefragments to form probe-fragment hybrids; and (c) detecting typable lociof the probe-fragment hybrids.

In an additional aspect, the present invention features a method ofamplifying genomic DNA, including the steps of providing isolated doublestranded genomic DNA, producing nicked DNA by contacting the doublestranded genomic DNA with a nicking agent, contacting this nicked DNAwith a strand displacing polymerase and a plurality of primers, so as toamplify the genomic DNA.

The invention further provides a method for detecting typable loci of agenome. The method includes the steps of (a) in vitro transcribing aplurality of amplified gDNA fragments, thereby obtaining genomic RNA(gRNA) fragments; (b) hybridizing the gRNA fragments with a plurality ofnucleic acid probes having sequences corresponding to the typable loci;and (c) detecting typable loci of the gRNA fragments that hybridize tothe probes.

The invention further provides a method of producing a reducedcomplexity, locus-specific, amplified representative population ofgenome fragments. The method includes the steps of (a) replicating anative genome with a plurality of random primers, thereby producing anamplified representative population of genome fragments; (b) replicatinga sub-population of the amplified representative population of genomefragments with a plurality of different locus-specific primers, therebyproducing a locus-specific, amplified representative population ofgenome fragments; and (c) isolating the sub-population, therebyproducing a reduced complexity, locus-specific, amplified representativepopulation of genome fragments.

The invention also provides a method for inhibiting ectopic extension ofprobes in a primer extension assay. The method includes the steps of (a)contacting a plurality of probe nucleic acids with a plurality of targetnucleic acids under conditions wherein probe-target hybrids are formed.;(b) contacting the plurality of probe nucleic acids with an ectopicextension inhibitor under conditions wherein probe-ectopic extensioninhibitor hybrids are formed; and (c) selectively modifying probes inthe probe-target hybrids compared to probes in the probe-ectopicextension inhibitor hybrids.

Further provided is a method including the steps of (a) contacting aplurality of genome fragments with a plurality of different immobilizednucleic acid probes under conditions wherein immobilized probe-fragmenthybrids are formed; (b) modifying the immobilized probes whilehybridized to the genome fragments, thereby forming modified immobilizedprobes; (c) removing said genome fragments from said probe-fragmenthybrids; and (d) detecting the modified immobilized probes afterremoving the genome fragments, thereby detecting typable loci of thegenome fragments.

The invention also provides a method including the steps of (a)representationally amplifying a native genome, wherein an amplifiedrepresentative population of genome fragments having the typable loci isproduced under isothermal conditions; (b) contacting the genomefragments with a plurality of nucleic acid probes having sequencescorresponding to the typable loci under conditions whereinprobe-fragment hybrids are formed; and (c) detecting typable loci of theprobe-fragment hybrids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of a whole genome genotyping (WGG) method of theinvention,

FIG. 2 shows exemplary probes useful for detection of typable loci usingallele-specific primer extension (ASPE) or single base extension (SBE).

FIG. 3 shows, in Panel A, agarose gels loaded with amplificationproducts from whole genome amplification reactions carried out undervarious conditions, and in Panel B, a table of yields calculated for thereactions.

FIG. 4 shows an image of an array signal from yeast genomic DNA assayedon a BeadArray™ (Panel A) and a subset of perfect match (PM) andmismatch. (MM) intensities for 18 loci out of 192 assayed from fourdifferent quadruplicate arrays (R5C1, R5C2, R6C1, R6C2) (Panel B). ThePM probes are the first set of four intensity values and MM probes arethe second set of four intensity values denoted by each probe type labelon the lower axis.

FIG. 5 shows array-based SBE genotyping performed on human gDNA directlyhybridized to BeadArrays™.

FIG. 6 shows array-based ASPE genotyping performed on human gDNAdirectly hybridized to a BeadArray™. Panel A shows raw intensity valuesacross the 77 probe pairs and Panel B shows the discrimination ratios(PM/PM+MM) plotted for the 77 loci.

FIG. 7 shows Genotyping scores of unamplified genomic DNA compared torandom primer amplified (RPA) genomic DNA using the GoldenGate™ assay(the amount of DNA input in the RPA reaction is shown below each bar,the RPA reactions employed random 9-mer oligonucleotides, except wherethe use of hexanucleotides (6-mer) or dodecanucleotides (12-mer) arespecified).

FIG. 8 shows a diagram of an exemplary method for generating genomic RNAas a target nucleic acid for amplification or detection.

FIG. 9 shows a diagram of an exemplary method for generating a reducedcomplexity, locus-specific representative population of genomefragments.

FIG. 10 shows an exemplary signal amplification scheme.

FIG. 11 shows, in Panel A, an image of a BeadArray™ hybridized withgenomic DNA fragments and detected with ASPE, and in Panel B, a GenTrainplot in which two homozygous (B/B and A/A) clusters and one heterozygous(A/B) cluster at one locus are differentiated.

FIG. 12 shows, in Panel A, a table of genotyping accuracy statistics; inPanels B and C GenCall plots for two samples (the line at 0.45 indicatesa lower threshold used to filter data to be called) and in Panels D andE, GenTrain plots for two loci (arrows indicate questionable data pointsthat were not called as they fell below a threshold of 0.45 in GenCallplots).

FIG. 13 shows diagrams illustrating ectopic extension (Panel A) andmethods for inhibiting ectopic extension including inhibition by bindingsingle-stranded probes to SSB (Panel B); blocking the 3′ end of theprobes with nucleic acids having complementary sequences (Panel C); andformation of unextendable hairpins (Panel D).

FIG. 14 shows scatter plots for Klenow-primed ASPE reactions onBeadArrays™ comparing assay signal in the presence and absence of singlestranded binding protein (SSB). The scatter plot in panel A shows theeffect of SSB on ectopic signal intensity in the absence of amplifiedgenomic DNA, whereas the scatter plot in panel B shows the effect of SSBon signal intensity in the presence of amplified genomic DNA. Panels Cand D show plots of the intensity for loci (sorted in order ofincreasing intensity) for either Klemnow (Panel C) or Klentaq (Panel D)ASPE reactions run on BeadArrays™ in the absence of an amplifiedpopulation of genome fragments (ntc-no target control provides a measureof “ectopic” extension).

FIGS. 15A-15C show scatter plots comparing intensity values for probesfollowing ASPE detection of populations of genome fragments produced byrandom primer amplification (amplified) and/or unamplified genomic DNA(unamplified).

FIGS. 16A-16B show a distribution of the number of probes (counts)having particular ratios of signal intensities for unamplifed toamplified DNA inputs (ratio of amplified:unamplified).

FIG. 17 shows exemplary genoplots for four loci (1824, 2706, 3633 and6126) detected from representationally amplified populations of genomefragments using the GoldenGate™ assay. Representationally amplifiedpopulations of genome fragments were separately produced from genomicDNA samples in the three different amounts indicated in the legend.Control data points were obtained for unamplified genomic DNA detectedunder the same conditions using the GoldenGate™ assay. Clusters forcontrol data points identified by the GenTrain algorithm are circled andthe number of data points in each cluster indicated below the x-axis.For the 2706 locus the empty cluster indicates a predicted clusterlocation for the AA genotype based on locations of the AB and BBclusters.

FIGS. 18A-18B show (A) a bar graph plotting the average intensitydetected for all probes on each array (LOD) following hybridization andASPE detection of RPA reaction mixtures generated from different amountsof input genomic DNA. (input) and (B) a bar graph plotting the ratio (PMsignal intensity/(PM signal intensity+MM signal intensity) for allprobes of an array (ratio) when used to probe RPA mixtures produced fromvarying amounts of input genornic DNA (input).

FIGS. 19A-19C show representative Genoplots for the 860 locus (panel A)and 954 locus (Panel B) for random primer amplified human genomefragments produced from 95 CEPH human samples and detected by allelespecific primer extension of probes on an array having probes specificfor the 1500 HapMap QC set of loci. Panel C shows the distribution ofloci according to genotype cluster separation score.

FIG. 20 shows signal intensity for perfect match (PM) and mismatch (MM)probes following allele-specific primer extension detection andtreatment with or without 0.1 N NaOH.

FIG. 21 shows (A) treatment of bisulfite-generated DNA fragments withalkaline phosphatase and T4 DNA kinase to generate either completelydephosphorylated or 3′ dephosphorylated products, respectively; (B)treatment of 3′ dephosphorylated DNA with T4 RNA ligase to produceconcatenated DNA followed by amplification in a strand-displacing, wholegenome, random primer amplification reaction; (C) treatment ofbisuifite-generated DNA fragments with terminal deoxynucleotidestransferase (TdT) and T4 RNA ligase to add universal tail sequences tothe fragments followed by PCR amplification; (D) treatment ofbisulfite-generated DNA fragments with T4 RNA ligase to add 5′ and 3′universal tail sequence tails to the bisulfite product followed by PCRamplification.

DEFINITIONS

As used herein, the term “genome” is intended to mean the fullcomplement of chromosomal DNA found within the nucleus of a eukaryoticcell. The term can also be used to refer to the entire geneticcomplement of a prokaryote, virus, mitochondrion or chloroplast or tothe haploid nuclear genetic complement of a eukaryotic species.

As used herein, the term “genomic DNA” or “gDNA” is intended to mean oneor more chromosomal polymeric deoxyribonucleotide molecules occurringnaturally in the nucleus of a eukaryotic cell or in a prokaryote, virus,mitochondrion or chloroplast and containing sequences that are naturallytranscribed into RNA as well as sequences that are not naturallytranscribed into RNA by the cell. A gDNA of a eukaryotic cell containsat least one centromere, two telomeres, one origin of replication, andone sequence that is not transcribed into RNA by the eukaryotic cellincluding, for example, an intron or transcription promoter. A gDNA of aprokaryotic cell contains at least one origin of replication and onesequence that is not transcribed into RNA by the prokaryotic cellincluding, for example, a transcription promoter. A eukaryotic genomicDNA can be distinguished from prokaryotic, viral or organellar genomicDNA, for example, according to the presence of introns in eukaryoticgenomic DNA and absence of introns in the gDNA of the others.

As used herein, the term “detecting” is intended to mean any method ofdetermining the presence of a particular molecule such as a nucleic acidhaving a specific nucleotide sequence. Techniques used to detect anucleic acid include, for example, hybridization to the sequence to bedetected. However, particular embodiments of this invention need notrequire hybridization directly to the sequence to be detected, butrather the hybridization can occur near the sequence to be detected, oradjacent to the sequence to be detected. Use of the term “near” is meantto imply within about 150 bases from the sequence to be detected. Otherdistances along a nucleic acid that are within about 150 bases andtherefore near include, for example, about 100, 50 40, 30, 20, 19, 18,17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 bases fromthe sequence to be detected. Hybridization can occur at sequences thatare further distances from a locus or sequence to be detected including,for example, a distance of about 250 bases, 500 bases, 1 kilobase ormore up to and including the length of the target nucleic acids orgenome fragments being detected.

Examples of reagents which are useful for detection include, but are notlimited to, radiolabeled probes, fluorophore-labeled probes, quantumdot-labeled probes, chromophore-labeled probes, enzyme-labeled probes,affinity ligand-labeled probes, electromagnetic spin labeled probes,heavy atom labeled probes, probes labeled with nanoparticle lightscattering labels or other nanoparticles or spherical shells, and probeslabeled with any other signal generating label known to those of skillin the art. Non-limiting examples of label moieties useful for detectionin the invention include, without limitation, suitable enzymes such ashorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; members of a binding pair that are capable offorming complexes such as streptavidin/biotin, avidin/biotin or anantigen/antibody complex including, for example, rabbit IgG andanti-rabbit IgG; fluorophores such as umbelliferone, fluorescein.,fluorescein isothiocyanate, rhodamine, tetramethyl rhodamine, eosin,green fluorescent protein, erythrosin, coumarin, methyl coumarin,pyrene, malachite green, stilbene, lucifer yellow, Cascade Blue™, TexasRed, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin,fluorescent lanthanide complexes such as those including Europium andTerbium, Cy3, Cy5, molecular beacons and fluorescent derivativesthereof, as well as others known in the art as described, for example,in Principles of Fluorescence Spectroscopy, Joseph R. Lakowicz (Editor),Plenum Pub Corp, 2nd edition (July 1999) and the 6^(th) Edition of theMolecular Probes Handbook by Richard P. Hoagland; a luminescent materialsuch as luminol; light scattering or plasmon resonant materials such asgold or silver particles or quantum dots; or radioactive materialinclude ¹⁴C, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, Tc99m, ³⁵S or ³H.

As used herein, the term “typable loci” is intended to meansequence-specific locations in a nucleic acid. The term can includepre-determined or predicted nucleic acid sequences expected to bepresent in isolated nucleic acid molecules. The term typable loci ismeant to encompass single nucleotide polymorphisms (SNPs), mutations,variable number of tandem repeats (VNTRs) and single tandem repeats(STRs), other polymorphisms, insertions, deletions, splice variants orany other known genetic markers. Exemplary resources that provide knownSNPs and other genetic variations include, but are not limited to, thedhSNP administered by the NOM and available online atncbi.nlm.nih.gov/SNP/ and the HCVBASE database described in Fredman etal. Nucleic Acids Research, 30:387-91, (2002) and available online athgvbase.cgb.ki.se/.

As used herein, the term “representationally amplifying” is intended tomean replicating a nucleic acid template to produce a nucleic acid copyin which the proportion of each sequence in the copy relative to allother sequences in the copy is substantially the same as the proportionsin the nucleic acid template. A nucleic acid template included in theterm can be a single molecule such as a chromosome or a plurality ofmolecules such as a collection of chromosomes making up a genome orportion of a genome. Similarly, a nucleic acid copy can be a singlemolecule or plurality of molecules. The nucleic acids can be DNA or RNAor mimetics or derivatives thereof. A copy nucleic acid can be aplurality of fragments that are smaller than the template DNA.Accordingly, the term can include replicating a genome, or portionthereof, such that the proportion of each resulting genome fragment toall other genome fragments in the population is substantially the sameas the proportion of its sequence to other genome fragment sequences inthe genome. The DNA being replicated can be isolated from a tissue orblood sample, from a forensic sample, from a formalin-fixed cell, orfrom other sources. A genomic DNA used in the invention can be intact,largely intact or fragmented. A nucleic acid molecule, such as atemplate or a copy thereof can be any of a variety of sizes including,without limitation, at most about 1 mb, 0.5 mb, 0.1 mb, 50 kb, 10 kb, 5kb, 3 kb, 2 kb, 1 kb, 0.5 kb, 0.25, 0.1. 0.05 or 0.02 kb.

Accordingly, the term “amplified representative” is intended to mean anucleic acid copy in which the proportion of each sequence in the copyrelative to all other sequences in the copy is substantially the same asthe proportions in the nucleic acid template. When used in reference toa population of genome fragments, for example, the term is intended tomean a population of genome fragments in which the proportion of eachgenome fragment to all other genome fragments in the population issubstantially the same as the proportion of its sequence to the othergenome fragment sequences in the genome. Substantial similarity betweenthe proportion of sequences in an amplified representation and atemplate genomic DNA means that at least 60% of the loci in therepresentation are no more than 5 fold over-represented orunder-represented. In such representations at least 70%, 80%, 90%, 95%or 99% of the loci can be, for example, no more than 5, 4, 3 or 2 foldover-represented or under-represented. A nucleic acid included in theterm can be DNA, RNA or an analog thereof. The number of copies of eachnucleic acid sequence in an amplified representative population can be,for example, at least 2, 5, 10, 25, 50, 100, 1000, 1×10⁴, 1×10⁵, 1×10⁶,1×10⁷, 1×10⁸ or 1×10¹⁰ fold more than the template or more.

Exemplary populations of genome fragments that include sequencesidentical to a portion of a genome include, for example, high complexityrepresentations or low complexity representations. As used herein, theterm “high complexity representation” is intended to mean a nucleic acidcopy having at least about 50% of the sequence of its template. Thus ahigh complexity representation of a genomic DNA can include, withoutlimitation at least about 60%, 70%, 75%, 80%, 85%, 90%, 95% or 99% ofthe template genome sequence. As used herein, the term “low complexityrepresentation” is intended to mean a nucleic acid copy having at mostabout 49% of the sequence of its template. Thus, a low complexityrepresentation of a genomic DNA can include, without limitation, at mostabout 49%, 40%, 30%, 20%, 10%, 5% or 1% of the genome sequence. Inparticular embodiments, a population of genome fragments of theinvention can have a complexity representing at least about 5%, 10%,20%, 30%, or 40% of the genome sequence.

As used herein, the term “directly detecting,” when used in reference toa. nucleic acid, is intended to mean perceiving or discerning a propertyof the nucleic acid in a sample based on the level of the nucleic acidin the sample. The term can include, for example, perceiving ordiscerning a property of a nucleic acid in a sample without amplifyingthe nucleic acid in the sample, or detection without amplification. Anexemplary property that can be perceived or discerned includes, withoutlimitation, a nucleotide sequence, the presence of a particularnucleotide such as a polymorphism or mutation at a particular site in asequence, or the like. One non-limiting example of a direct detectionmethod is the detection of a nucleic acid by hybridizing a labeled probeto the nucleic acid and determining the presence of the nucleic acidbased on presence of the hybridized label. Other examples of directdetection are described herein and include, for example, single baseextension (SBE) and allele-specific primer extension (ASPE), Thoseskilled in the art will understand that following detection, a sample ofunamplified nucleic acid, such as a sample of unamplified genomic DNAfragments, can be amplified.

In particular embodiments, direct detection can include generating adouble-stranded nucleic acid complex between a typable locus and itscomplementary sequence and perceiving the complex without generatingadditional copies of the typable locus, In some embodiments, directdetection of a typable locus can involve formation of a singlehybridization complex thereby excluding repeated hybridization to aparticular nucleic acid molecule having the typable

A method of detecting a detectable position, such as a typable locus orsequence genetically linked to a typable locus can include, for example,hybridization by an oligonucleotide to the interrogation position, orhybridization by an oligonucleotide nearby or adjacent to theinterrogation position, followed by extension of the hybridizedoligonucleotide across the interrogation position.

Several direct detection methods useful in the invention and describedherein, including, without limitation, SBE and ASPE, employ probes thatboth capture a genome fragment and produce a signal indicative of thepresence of a particular SNP locus on the fragment. In particular, amethod of the invention can be carried out under conditions in whichdetection of a SNP or other feature of a captured oligonucleotide, suchas a genome fragment, does not require an exogenously added queryoligonucleotide. However, if desired, exogenously added queryoligonucleotides can be used. Exemplary methods employing exogenouslyadded query oligonucleotides are set forth below such as oligo ligationassay (OLA), extension ligation (GoldenGate™), rolling circle-baseddetection methods, allele-specific oligonucleotide (ASO) hybridizationand others.

As used herein, the term “amplify,” when used in reference to a singlestranded nucleic acid, is intended to mean producing one or more copiesof the single stranded nucleic acid, or a portion thereof.

As used herein, the term “genome fragment” is intended to mean anisolated nucleic acid molecule having a sequence that is substantiallyidentical to a portion of a chromosome. A chromosome is understood to bea linear or sometimes circular DNA-containing body of a virus,prokaryotic organism, or eukaryotic nucleus that contains most or all ofthe replicated genes. A population of genome fragments can includesequences identical to substantially an entire genome or a portionthereof. A genome fragment can have, for example, a sequence that issubstantially identical to at least about 25, 50, 70, 100, 200, 300,400, 500, 600, 700, 800, 900 or 1000 or more nucleotides of achromosome. A genome fragment can be DNA, RNA, or an analog thereof. Itwill be understood by those skilled in the art that an RNA sequence andDNA chromosome sequence that differ by the presence of uracils in placeof thymines are substantially identical in sequence.

As used herein, the term “native,” when used in reference to a genome,is intended to mean produced by isolation fro a cell or other host. Theterm is intended to exclude genomes that are produced by in vitrosynthesis, replication or amplification.

As used herein, the term “corresponding to,” when used in reference to atypable locus, is intended to mean having a nucleotide sequence that isidentical or complimentary to the sequence of the typable locus, or adiagnostic portion thereof. Exemplary diagnostic portions include, forexample, nucleic acid sequences adjacent or near to the typable locus ofinterest.

As used herein, the term “multiplex” is intended to mean simultaneouslyconducting a plurality of assays on one or more sample. Multiplexing canfurther include simultaneously conducting a plurality of assays in eachof a plurality of separate samples, For example, the number of reactionmixtures analyzed can be based on the number of wells in a multi-wellplate and the number of assays conducted in each well can be based onthe number of probes that contact the contents of each well. Thus, 96well, 384 well or 1536 well microliter plates will utilize compositearrays comprising 96, 384 and 1536 individual arrays, although as willbe appreciated by those in the art, not each microliter well needcontain an individual array. Depending on the size of the microtiterplate and the size of the individual array, very high numbers of assayscan be run simultaneously; for example, using individual arrays of 2,000and a 96 well microliter plate, 192,000 experiments can be done at once;the same arrays in a 384 microliter plate yields 768,000 simultaneousexperiments, and a 1536 microliter plate gives 3,072,000 experiments.Although multiplexing has been exemplified with respect to microliterplates, it will be understood that other formats can be used formultiplexing including, for example, those described in US 2002/0102578A1.

As used herein, the term “polymerase” is intended to mean an enzyme thatproduces a complementary replicate of a nucleic acid molecule using thenucleic acid as a template strand. DNA polymerases bind to the templatestrand and then move down the template strand adding nucleotides to thefree hydroxyl group at the 3′ end of a growing chain of nucleic acid.DNA polymerases synthesize complementary DNA molecules from DNA or RNAtemplates and RNA polymerases synthesize RNA molecules from DNAtemplates (transcription). DNA polymerases generally use a short,preexisting RNA or DNA strand, called a primer, to begin chain growth.Some DNA polymerases can only replicate single-stranded templates, whileother DNA polymerases displace the strand upstream of the site wherethey are adding bases to a chain. As used herein, the term “stranddisplacing,” when used in reference to a. polymerase, is intended tomean having an activity that removes a complementary strand from atemplate strand being read by the polymerase. Exemplary polymeraseshaving strand displacing activity include, without limitation the largefragment of list (Bacillus stearothermophilus) polymerase, exo⁻ Klenowpolymerase or sequencing grade T7 exo-polymerase.

Further, some DNA polymerases degrade the strand in front of them,effectively replacing it with the growing chain behind. This is known asan exonuclease activity. Some DNA polymerases in use commercially or inthe lab have been modified, either by mutation or otherwise, to reduceor eliminate exonuclease activity. Further mutations or modification arealso frequently performed to improve the ability of the DNA polymeraseto use non-natural nucleotides as substrates.

As used herein, the term “processivity” refers to the number of bases,on average, added to a nucleic acid being synthesized by a polymeraseprior to the polymerase detaching from the template nucleic acid beingreplicated. Polymerases of low processivity, on average, synthesizeshorter nucleic acid chains compared to polymerases of highprocessivity. A polymerase of low processivity will synthesize, on theaverage, a nucleic acid that is less than about 100 bases in lengthprior to detaching from the template nucleic acid being replicated.Further exemplary average lengths for a nucleic acid synthesized by alow processivity polymerase prior to detaching from the template nucleicacid being replicated include, without limitation, less than about 80,50, 25, 10 or 5 bases.

As used herein, the term “nicked,” when used in reference to adouble-stranded nucleic acid, is intended to mean lacking at least onecovalent bond of the backbone connecting adjacent sequences in a firststrand and having a complimentary second strand hybridized to both ofthe adjacent sequences in the first strand.

As used herein, the term “nicking agent” is intended to mean a physical,chemical, or biochemical entity that cleaves a covalent bond connectingadjacent sequences in a first nucleic acid strand, thereby producing aproduct in which the adjacent sequences are hybridized to the samecomplementary strand. Exemplary nicking agents include, withoutlimitation, single strand nicking restriction endonucleases thatrecognize a specific sequence such as N.BstNBI, MutH or genell proteinof bacteriophage fl; DNAse I; chemical reagents such as free radicals;or ultrasound.

As used herein, the term “isolated,” when used in reference to abiological substance, is intended to mean removed from at least aportion of the molecules associated with or occurring with the substancein its native environment. Accordingly, the term “isolating,” when usedin reference to a biological substance, is intended to mean removing thesubstance from its native environment or removing at least a portion ofthe molecules associated with or occurring with the nucleic acid orsubstance in its native environment. Exemplary substances that can beisolated include, without limitation, nucleic acids, proteins,chromosomes, cells, tissues or the like. An isolated biologicalsubstance, such as a nucleic acid, can be essentially free of otherbiological substances. For example, an isolated nucleic acid can be atleast about 90%, 95%, 99% or 100% free of non-nucleotide materialnaturally associated with it. An isolated nucleic acid can, for example,be essentially free of other nucleic acids such that its sequence isincreased to a significantly higher fraction of the total nucleic acidpresent in the solution of interest than in the cells from which thesequence was taken. For example, an isolated nucleic acid can be presentat a 2, 5, 10, 50, 100 or 1000 fold or higher level than other nucleicacids in vitro relative to the levels in the cells from which it wastaken. This could be caused by preferential reduction in the amount ofother DNA or RNA present, or by a preferential increase in the amount ofthe specific DNA or RNA sequence, or by a combination of the two.

As used herein, the term “complexity,” when used in reference to anucleic acid sequence, is intended to mean the total length of uniquesequence in a genome. The complexity of a genome can be equivalent to orless than the length of a single copy of the genome (i.e. the haploidsequence). Estimates of genome complexity can be less than the totallength if adjusted for the presence of repeated sequences. The length ofrepeated sequences used for such estimates can be adjusted to suit aparticular analysis. For example, complexity can be the sum of thenumber of unique sequence words in a haploid genome sequence plus thelength of the sequence word. A sequence word is a continuous sequence ofa defined length of at least 10 nucleotides. The number of repeatsequences, and thus, the length of unique sequence, in a genome willdepend. upon the length of the sequence word. More specifically, as thelength of the sequence word is increased to, for example, 15, 20, 25,30, 50, 100 or more nucleotides, the complexity estimate will generallyincrease approaching the upper limit of the length of the haplotypegenome.

DETAILED DESCRIPTION OF TUE INVENTION

One object of the invention is to provide a sensitive and accuratemethod for simultaneously interrogating a plurality of gene loci in aDNA sample. In particular, a method of the invention can be used todetermine the genotype of an individual by direct detection of aplurality of single nucleotide polymorphisms in a sample of theindividual's genomic DNA or cDNA. An advantage of the invention is thata small amount of genomic DNA can be obtained from an individual, andamplified to obtain an amplified representative population of genomefragments that can be interrogated in the methods of the invention.Thus, the methods are particularly useful for genotyping genomic DNAobtained from relatively small tissue samples such as a biopsy orarchived sample. Generally, the methods will be used to amplify arelatively small number of template genome copies. In particularembodiments, a genomic DNA sample can be obtained from a single cell andgenotyped.

A further advantage of direct detection of genetic loci in the methodsof the invention is that a target genomic DNA fragment need not beamplified once it has been captured by an appropriate probe. Thus, themethods can provide the advantage of reducing or obviating the need forelaborate and expensive means for detection following capture. Ifsufficient DNA is present, the detection of typable loci can beconducted by a technique that does not require amplification of acaptured target such as single base extension (SBE) or allele specificprimer extension (ASPE). Other methods of direct detection includeligation, extension-ligation, invader assay, hybridization with alabeled complementary sequence, or the like. Such direct detectiontechniques can be carried out, for example, directly on a capturedprobe-target complex as set forth below. Although targetamplification-based detection methods are not required in the methods ofthe invention, the methods are compatible with a variety ofamplification based detection methods such as Invader, PCR-based, oroligonucleotide ligation assay-based (OLA-based) technologies which canbe used, if desired.

The invention provides methods of whole genome amplification that can beused to amplify genomic DNA prior to genetic evaluation such asdetection of typable loci in the genome. Whole genome amplificationmethods of the invention can be used to increase the quantity of genomicDNA without compromising the quality or the representation of any givensequence. Thus, the methods can be used to amplify a relatively smallquantity of genomic DNA in a sequence independent fashion to providelevels of the gnomic DNA that can be genotyped, Surprisingly, a complexgnome can be amplified with a low processivity polymerase to obtain apopulation of genome fragments that is representative of the genome, hashigh complexity and contains fragments that have a convenient size forhybridization to a typical nucleic acid array.

As set forth in further detail below, a complex representativepopulation of genome fragments can be incubated with a plurality ofprobes and a relatively small fraction of these fragments, having lociof interest, specifically detected despite the presence substantiallylarge amount of other genomic sequences present in the population offragments. Moreover, specific detection can occur for such complexrepresentations even if probe hybridization is carried out with largeamounts and high concentrations of the genome fragment populations.Thus, an advantage of the invention is that whole genome genotyping canbe carried out in the presence of a high complexity genomic DNAbackground.

Furthermore, amplification of genomic DNA in the methods disclosedherein does not require the polymerase chain reaction. Specifically,amplification can be carried out such that sequences are amplifiedseveral fold under isothermal conditions. Thus, although an elevatedtemperature step can be used, for example, to initially denature agenomic DNA template, temperature cycling need not be used. Accordingly,repeated increases in temperature, normally used to denature hybrids,and repeated return to hybridization temperatures need not be used.

After capture and separation of the typable loci on an array, theindividual typable loci can be scored in positus (in place) via asubsequent detection assay such as ASPE or SBE, Thus, a population ofgenome fragments obtained by whole genome amplification with a lowprocessivity polymerase can be captured by an array of probes and thegenotype of the genome determined based on the typable loci detectedindividually at each probe as set forth below and demonstrated in theExamples. An in positus genotyping approach has remarkable advantages inthat it allows extensive multiplexing of the assay where desired.

The use of high density DNA array technology for detection of typableloci in a whole genome or complex DNA sample, such as a cDNA sample, canbe facilitated by the amplification methods of the invention because themethod can produce a number of copies of typable loci, or sequencescomplementary to typable loci to scale in relative proportion to theirrepresentation in the template sample. Maintaining relatively uniformrepresentation is advantageous in many applications because if someareas of the genome containing specific genetic markers are notfaithfully replicated, they will not be detected in an assay adjustedfor the average amplification. The invention can by scaled to detect adesired number of typable loci simultaneously or sequentially asdesired. The methods can be used to simultaneously detect at least 10typable loci, at least 100, 1000, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷ typableloci or more. Similarly, these numbers of typable loci can be determinedin a sequential format where desired, Thus, the invention can be used togenotype individuals on a genome-wide scale if desired.

The whole genome amplification methods of the invention and whole genomegenotyping methods of the invention are useful, alone or in combination,in a number of applications including, for example, single cell spermhaplotype analysis, genotyping of large numbers of individuals in ahigh-throughput format, or identification of new haplotypes.Furthermore, the invention reduces the amount of DNA or RNA samplerequired in many current array assays. Further still, improved arraysensitivity available with the invention can lead to reduced samplerequirements, improved LOD scoring ability, and greater dynamic range.

The invention can be used to identify new markers or haplotypes that arediagnostic of traits such as those listed above. Such studies can becarried out by comparing genotypes for groups of individuals having ashared trait or set of traits with a control group lacking the traitbased on the expectation that there will be higher frequencies of thecontributing genetic components in a group of people with a sharedtrait, such as a particular disease or response to a drug, vaccine,pathogen, or environmental factor, than in a group of similar peoplewithout the disease or response. Accordingly the methods of theinvention can be used to find chromosome regions that have differenthaplotype distributions in the two groups of people, those with adisease or response and those without. Each region can then be studiedin more detail to discover which variants in which genes in the regioncontribute to the disease or response, leading to more effectiveinterventions. This can also allow the development of tests to predictwhich drugs or vaccines are effective in individuals with particulargenotypes for genes affecting drug metabolism. Thus, the invention canbe used to determine the genotype of an individual based onidentification of which genetic markers are found in the individual'sgenome. Knowledge of an individual's genotype can be used to determine avariety of traits such as response to environmental factors,susceptibility to infection, effectiveness of particular drugs orvaccines or risk of adverse responses to drugs or vaccines.

The invention is exemplified herein with respect to amplification and/ordetection of typable loci for a whole genome. Those skilled in the artwill recognize from the teaching herein that the methods can also beused with other complex nucleic acid samples including, for example, afraction of a genome, such as a chromosome or subset of chromosomes; asample having multiple different genomes, such as a biopsy sample havinggenomic DNA from a host as well as one or more parasite or an ecologicalsample having multiple organisms from a particular environment; or evencDNA or an amplified cDNA representation. Accordingly, the methods canbe used to characterize typable loci found in a fraction of a genome orin a mixed genome sample. The invention provides a method of detectingone or several typable loci contained within a given genome. The methodincludes the steps of (a) providing an amplified representativepopulation of genome fragments having such typable loci; (b) contactingthe genome fragments with a plurality of nucleic acid probes havingsequences corresponding to the typable loci under conditions whereinprobe-fragment hybrids are formed; and (c) detecting typable loci of theprobe-fragment hybrids. In particular embodiments these nucleic acidprobes are at most 125 nucleotides in length. FIG. 1 shows a generaloverview of an exemplary method of detecting typable loci of a genome.As shown in FIG. 1 , a population of genome fragments can be obtainedfrom a genome, denatured and contacted with an array of nucleic acidprobes each having a sequence that is complementary to a particulartypable locus of the genome. Genome fragments having typable locirepresented on the probes are captured as probe-fragment hybrids atdiscrete locations on the array while other fragments lacking loci ofinterest will remain in bulk solution. The probe-fragment hybrids can bedetected by enzyme-mediated addition of a detection moiety (referred toas a signal moiety in FIG. 1 ) to the probe. In the exemplary embodimentof FIG. 1 , a polymerase selectively adds a biotin labeled nucleotide toprobes in probe-fragment hybrids. The biotinylated probes can then bedetected, for example, by contacting a fluorescently labeled avidin tothe array under conditions where biotinylated probes are selectivelybound and detecting the locations in the array that fluoresce. Based onthe known sequences for probes at each location, the presence ofparticular typable loci can be determined.

A method of the invention can be used to amplify genomic DNA (gDNA) ordetect typable loci of a genome from any organism. The methods areideally suited to the amplification and analysis of large genomes suchas those typically found in eukaryotic unicellular and multicellularorganisms. Exemplary eukaryotic gDNA that can be used in a method of theinvention includes, without limitation, that from a mammal such as arodent, mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig,goat, cow, cat, dog, primate, human or non-human primate; a plant suchas Arabidopsis thaliana, corn (Zea mays), sorghum, oat (Oryza saliva),wheat, rice, canola, or soybean; an algae such as Chlamydomonasreinhardtii; a nematode such as Caenorhabditis elegans; an insect suchas Drosophila melanogaster, mosquito, fruit fly, honey bee or spider; afish such as zebrafish (Danio rerio); a reptile; an amphibian such as afrog or Xenopus laveis; a Dictyasielizan discoideum; a fungi such asPneumocystis carinii, Takifugu rubripes, yeast, Saccharamoycescerevisiae or Schizosaccharomyces pombe; or a Plasmodium falciparum. Amethod of the invention can also be used to detect typable loci ofsmaller genomes such as those from a prokaryote such as a bacterium,Escherichia coli, staphylococci or Mycoplasma pneumoniae; an archae; avirus such as Hepatitis C virus or human immunodeficiency virus; or aviroid.

A genomic DNA used in the invention can have one or more chromosomes.For example, a prokaryotic genomic DNA including one chromosome can beused. Alternatively, a eukaryotic genomic DNA including a plurality ofchromosomes can be used in a method of the invention. Thus, the methodscan be used, for example, to amplify or detect typable loci of a genomicDNA having n equal to 2 or more, 4 or more, 6 or more, 8 or more, 10 ormore, 15 or more, 20 or more, 23 or more, 25 or more, 30 or more, or 35or more chromosomes, where n is the haploid chromosome number and thediploid chromosome count is 2n The size of a genomic DNA used in amethod of the invention can also be measured according to the number ofbase pairs or nucleotide length of the chromosome complement. Exemplarysize estimates for some of the genomes that are useful in the inventionare about 3.1 Gbp (human), 2.7 Gbp (mouse), 2.8 Gbp (rat), 1.7 Gbp(zebrafish), 165 Mbp (fruitfly), 13.5 Mbp (S. cerevisiae), 390 Mbp(fugu), 278 Mbp (mosquito) or 103 Mbp (C. elegans). Those skilled in theart will recognize that genomes having sizes other than thoseexemplified above including, for example, smaller or larger genomes, canbe used in a method of the invention.

Genomic DNA can be isolated from one or more cells, bodily fluids ortissues. Known methods can be used to obtain a bodily fluid such asblood, sweat, tears, lymph, urine, saliva, semen, cerebrospinal fluid,feces or amniotic fluid. Similarly known biopsy methods can be used toobtain cells or tissues such as buccal swab, mouthwash, surgicalremoval, biopsy aspiration or the like. Genomic DNA can also be obtainedfrom one or more cell or tissue in primary culture, in a propagated cellline, a fixed archival sample, forensic sample or archeological sample.

Exemplary cell types from which gDNA can be obtained in a method of theinvention include, without limitation, a blood cell such as a Blymphocyte, T lymphocyte, leukocyte, erythrocyte, macrophage, orneutrophil; a muscle cell such as a skeletal cell, smooth muscle cell orcardiac muscle cell; germ cell such as a sperm or egg; epithelial cell;connective tissue cell such as an adipocyte, fibroblast or osteoblast;neuron; astrocyte; stromal cell; kidney cell; pancreatic cell; livercell; or keratinocyte. A cell from which gDNA is obtained can be at aparticular developmental level including, for example, a hematopoieticstem cell or a cell that arises from a hematopoietic stem cell such as ared blood cell, B lymphocyte, T lymphocyte, natural killer cellneutrophil, basophil, eosinophil, monocyte, macrophage, or platelet.Other cells include a bone marrow stromal cell (mesenchymal stem cell)or a cell that develops therefrom such as a bone cell (osteocyte),cartilage cells (chondrocyte), fat cell (adipocyte), or other kinds ofconnective tissue cells such as one found in tendons; neural stem cellor a cell it gives rise to including, for example, a nerve cells(neuron), astrocyte or oligodendrocyte; epithelial stem cell or a cellthat arises from an epithelial stem cell such as an absorptive cell,goblet cell, Paneth cell, or enteroendocrine cell; skin stem cell;epidermal stem cell; or follicular stem cell. Generally any type of stemcell can be used including, without limitation, an embryonic stem cell,adult stem cell, or pluripotent stem cell.

A cell from which a gDNA sample is obtained for use in the invention canbe a normal cell or a cell displaying one or more symptom of aparticular disease or condition. Thus, a gDNA used in a method of theinvention can be obtained from a cancer cell, neoplastic cell, necroticcell or the like. Those skilled in the art will know or be able toreadily determine methods for isolating gDNA from a cell, fluid ortissue using methods known in the art such as those described inSambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition,Cold Spring Harbor Laboratory, New York (2001) or in Ausubel et al.,Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore,Md. (1998). A method of the invention can further include steps ofisolating a particular type of cell or tissue. Exemplary methods thatcan be used in a method of the invention to isolate a particular cellfrom other cells in a population include, but are not limited to,Fluorescent Activated Cell Sorting (FACS) as described, for example, inShapiro, Practical Flow Cytometry, 3rd edition Wiley-Liss; (1995),density gradient centrifugation, or manual separation usingmicromanipulation methods with microscope assistance. Exemplary cellseparation devices that are useful in the invention include, withoutlimitation, a Beckman JE-6 centrifugal elutriation system, BeckmanCoulter EPICS ALTRA computer-controlled Flow Cytometer-cell sorter,Modular Flow Cytometer from Cytotnation, Inc., Coulter counter andchannelyzer system, density gradient apparatus, cytocentrifuge, BeckmanJ-6 centrifuge, EPICS V dual laser cell sorter, or EPICS PROFILE flowcytometer. A tissue or population of cells can also be removed bysurgical techniques. For example, a tumor or cells from a tumor can beremoved from a tissue by surgical methods, or conversely non-cancerouscells can be removed from the vicinity of a tumor. Using methods such asthose set forth in further detail below, the invention can be used tocompare typable loci for different cells including, for example,cancerous and non-cancerous cells isolated from the same individual orfrom different individuals.

A gDNA can be prepared for use in a method of the invention by lysing acell that contains the DNA. Typically, a cell is lysed under conditionsthat substantially preserve the integrity of the cell's gDNA. Inparticular, exposure of a cell to alkaline pH can be used to lyse a cellin a method of the invention while causing relatively little damage togDNA. Any of a variety of basic compounds can be used for lysisincluding, for example, potassium hydroxide, sodium hydroxide, and thelike. Additionally, relatively undamaged gDNA can be obtained from acell lysed by an enzyme that degrades the cell wall. Cells lacking acell wall either naturally or due to enzymatic removal can also be lysedby exposure to osmotic stress. Other conditions that can be used to lysea cell include exposure to detergents, mechanical disruption, sonicationheat, pressure differential such as in a French press device, or Douncehomogenization. Agents that stabilize gDNA can be included in a celllysate or isolated gDNA sample including, for example, nucleaseinhibitors, chelating agents, salts buffers and the like. Methods forlysing a cell to obtain gDNA can be carried out under conditions knownin the art as described, for example, in Sambrook et al., supra (2001)or in Ausubel et al., supra, (1998).

In particular embodiments of the invention, a crude cell lysatecontaining g can be directly amplified or detected without furtherisolation of the gDNA. Alternatively, a gDNA can be further isolatedfrom other cellular components prior to amplification or detection.Accordingly, a detection or amplification method of the invention can becarried out on purified or partially purified gDNA. Genomic DNA can beisolated using known methods including, for example, liquid phaseextraction, precipitation, solid phase extraction, chromatography andthe like. Such methods are often referred to as minipreps and aredescribed for example in Sambrook et al., supra, (2001) or in Ausubel etal., supra. (1998) or available from various commercial vendorsincluding, for example, Qiagen (Valencia, Calif.) or Promega (Madison,Wis.).

An amplified representative population of genome fragments can beprovided by amplifying a native genome under conditions that replicate agenomic DNA (gDNA) template to produce one or more copies in which therelative proportion of each copied sequence is substantially the same asits proportion in the original gDNA. Thus, a method of the invention caninclude a step of representationally amplifying a native genome. Any ofa variety of methods that replicate genomic .DNA in a sequenceindependent fashion can be used in the invention.

A method of the invention can be used to produce an amplifiedrepresentative population of genome fragments from a small number ofgenome copies. Accordingly, small tissue samples or other samples havingrelatively few cells, for example, due to low abundance, biopsyconstraints or high cost, can be genotyped or evaluated on a genome-widescale. The invention can be used to produce an amplified representativepopulation of genome fragments from a single native genome copyobtained, for example, from a single cell. In other exemplaryembodiments of the invention, an amplified representative population ofgenome fragments can be produced from larger number of copies of anative genome including, but not limited to, about 1,000 copies (for ahuman genome, approximately 3 nanograms of DNA) or fewer, 10,000 copiesor fewer, 1×10⁵ copies (for a human genome, approximately 300 nanogramsof DNA) or fewer, 5×10⁵ copies or fewer, 1×10⁶ copies or fewer, 1×10⁸copies or fewer, 1×10¹⁰ copies or fewer, or 1×10¹² copies or fewer.

A DNA sample that is representationally amplified in the invention canbe a genome such as those set forth above or other .DNA templates suchas mitochondrial DNA or some subset of genomic DNA. One non-limitingexample of a subset of genomic DNA is one particular chromosome or oneregion of a particular chromosome. In general, an amplification methodused in the invention can be carried out using at least one primernucleic acid that hybridizes to a template nucleic acid to form ahybridization complex, nucleotide triphosphates (NTPs) and a polymerasewhich modifies the primer by reacting the NTPs with the 3′ hydroxyl ofthe primer thereby replicating at least a portion of the template. Forexample, PCR based methods generally utilize a DNA template, twoprimers, dNTPs and a DNA polymerase. Thus, in a typical whole genomeamplification method of the invention, a genomic DNA sample is incubatedwith a reaction mixture that includes amplification components such asthose set forth above, and an amplified representative population ofgenome fragments is formed.

A primer used in a method of the invention can have any of a variety ofcompositions or sizes, so long as it has the ability to hybridize to atemplate nucleic acid with sequence specificity and can participate inreplication of the template. For example, a primer can be a nucleic acidhaving a native structure or an analog thereof. A nucleic acid with anative structure generally has a backbone containing phosphodiesterbonds and can be, for example, deoxyribonucleic acid or ribonucleicacid. An analog structure can have an alternate backbone including,without limitation, phosphoramide (see, for example, Beaucage et al.,Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J.Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579(1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al,Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470(1988); and Pauwels et at, Chemica Scripta 26:141 91986)),phosphorothioate (see, for example, Mag et at, Nucleic Acids Res.19:1437 (1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (see,for example, Briu et al., J. Am. Chem. Soc, 11 1:2321 (1989),O-methylphophoroamidite linkages (see, for example, Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see, forexample, Egholm. J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem.Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlssonet al., Nature 380:207 (1996)). Other analog structures include thosewith positive backbones (see, for example, Denpcy et al., Proc. Natl.Acad. Sci. USA 92:6097 (1995); non-ionic backbones (see, for example,U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863;Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1990;Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al.,Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASC SymposiumSeries 580, “Carbohydrate Modifications in Antisense Research”, Ed. Y.S. Sanghui and P. Dan Cook; Mesmacker et al., Bioorganic & MedicinalChem. Left. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17(1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones,including, for example, those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, “CarbohydrateModifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook.Analog structures containing one or more carbocyclic sugars are alsouseful in the methods and are described, for example, in Jenkins et al.,Chem. Soc. Rev. (1995) pp 169-176. Several other analog structures thatare useful in the invention are described in Rawls, C & F. News Jun. 2,1997 page 35.

A further example of a nucleic acid with an analog structure that isuseful in the invention is a peptide nucleic acid (PNA). The backbone ofa PNA is substantially non-ionic under neutral conditions, in contrastto the highly charged phosphodiester backbone of naturally occurringnucleic acids. This provides two non-limiting advantages. First, the PNAbackbone exhibits improved hybridization kinetics. Secondly, PNAs havelarger changes in the melting temperature (T_(a)) for mismatched versusperfectly matched base pairs. DNA and RNA typically exhibit a 2-4° C.drop in T_(m) for an internal mismatch. With the non-ionic PNA backbone,the drop is closer to 7-9° C. This can provide for better sequencediscrimination. Similarly, due to their non-ionic nature, hybridizationof the bases attached to these backbones is relatively insensitive tosalt concentration.

A nucleic acid useful in the invention can contain a non-natural sugarmoiety in the backbone. Exemplary sugar modifications include but arenot limited to 2′ modifications such as addition of halogen, alkyl,substituted alkyl, alicaryl, arallcyl, O-allcaryl or O-aralkyl, SH,SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2 CH3, ONO2, NO2, N3, NH2,heterocycloallcyl, heterocycloallcaryl, aminoallcylamino,polyallcylamino, substituted silyl, and the like. Similar modificationscan also be made at other positions on the sugar, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.

A nucleic acid used in the invention can also include native ornon-native bases. In this regard a native deoxyribonucleic acid can haveone or more bases selected from the group consisting of adenine,thymine, cytosine or guanine and a ribonucleic acid can have one or morebases selected from the group consisting of uracil, adenine, cytosine orguanine. Exemplary non-native bases that can be included in a nucleicacid, whether having a native backbone or analog structure, include,without limitation, inosine, xathanine, hypoxathanine, isocytosine,isoguanine, 5-methylcytosine, 5-hydroxymethyl cytosine, 2-aminoadenine,6-methyl adenine, 6-methyl guanine, 2-propyl guanine, 2-propyl adenine,2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil,15-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil,6-azo cytosine, 6-azo thymine, 4-thiouracil, 8-halo adenine or guanine,8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyladenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituteduracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine,8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine,3-deazaadenine or the like. A particular embodiment can utilizeisocytosine and isoguanine in a nucleic acid in order to reducenon-specific hybridization, as generally described in U.S. Pat. No.5,681,702.

A non-native base used in a nucleic acid of the invention can haveuniversal base pairing activity, wherein it is capable of base pairingwith any other naturally occurring base. Exemplary bases havinguniversal base pairing activity include 3-nitropyrrole and5-nitroindole. Other bases that can be used include those that have basepairing activity with a subset of the naturally occurring bases such asinosine which base pairs with cytosine, adenine or uracil.

A nucleic acid having a modified or analog structure can be used in theinvention, for example, to facilitate the addition of labels, or toincrease the stability or half-life of the molecule under amplificationconditions or other conditions used in accordance with the invention. Aswill be appreciated by those skilled in the art, one or more of theabove-described nucleic acids can be used in the present invention,including, for example, as a mixture including molecules with native oranalog structures. In addition, a nucleic acid primer used in theinvention can have a structure desired for a particular amplificationtechnique used in the invention such as those set forth below.

In particular embodiments a nucleic acid useful in the invention caninclude a detection moiety. A detection moiety can be used, for example,to detect one or more members of an amplified representative populationof genome fragments using methods such as those set forth below. Adetection moiety can be a primary label that is directly detectable orsecondary label that can be indirectly detected, for example, via director indirect interaction with a primary label. Exemplary primary labelsinclude, without limitation, an isotopic label such as a naturallynon-abundant radioactive or heavy isotope; chromophore; luminophore;fluorophore; calorimetric agent; magnetic substance; electron-richmaterial such as a metal; electrochemiluminescent label such as Ru(bpy)₃²⁺; or moiety that can be detected based on a nuclear magnetic,paramagnetic, electrical, charge to mass, or thermal characteristic.Fluorophores that are useful in the invention include, for example,fluorescent lanthanide complexes, including those of Europium andTerbium, fluorescein, rhodamine, tetramethylrhodamine, eosin,erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, Cy3,Cy5, stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, alexa dyes,phycoerythin, bodipy, and. others known in the art such as thosedescribed in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6thEdition; The Synthegen catalog (Houston, Tex.), Lakowicz, Principles ofFluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), or WO98/59066. Labels can also include enzymes such as horseradish peroxidaseor alkaline phosphatase or particles such as magnetic particles oroptically encoded nanoparticles.

Exemplary secondary labels are binding moieties. A binding moiety can beattached to a nucleic acid to allow detection or isolation of thenucleic acid via specific affinity for a receptor. Specific affinitybetween two binding partners is understood to mean preferential bindingof one partner to another compared to binding of the partner to othercomponents or contaminants in the system. Binding partners that arespecifically bound typically remain bound under the detection orseparation conditions described herein, including wash steps to removenon-specific binding. Depending upon the particular binding conditionsused, the dissociation constants of the pair can be, for example, lessthan about 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, or 10⁻¹²M⁻¹.

Exemplary pairs of binding moieties and receptors that can be used inthe invention include, without limitation, antigen and immunoglobulin oractive fragments thereof, such as FAbs; immunoglobulin andimmunoglobulin (or active fragments, respectively); avidin and biotin,or analogs thereof having specificity for avidin such as imino-biotin;streptavidin and biotin, or analogs thereof having specificity forstreptavidin such as imino-biotin; carbohydrates and lectins; and otherknown proteins and their ligands. It will be understood that eitherpartner in the above-described pairs can be attached to a nucleic acidand detected or isolated based on binding to the respective partner. Itwill be further understood that several moieties that can be attached toa nucleic acid can function as both primary and secondary labels in amethod of the invention. For example, strepatvidin-phycoerythrin can bedetected as a primary label due to fluorescence from the phycoerythrinmoiety or it can be detected as a secondary label due to its affinityfor anti-streptavidin antibodies, as set forth in further detail belowin regard to signal amplification methods.

In a particular embodiment, the secondary label can be a chemicallymodifiable moiety. In this embodiment, labels having reactive functionalgroups can be incorporated into a nucleic acid. The functional group canbe subsequently covalently reacted with a primary label. Suitablefunctional groups include, but are not limited to, amino groups, carboxygroups, maleimide groups, oxo groups and thiol groups. Binding moietiescan be particularly useful when attached to primers used foramplification of a gDNA because an amplified representative populationof genome fragments produced with such primers can be attached to anarray via said binding moieties. Furthermore, binding moieties can beuseful for separating amplified fragments from other components of anamplification reaction, concentrating the amplified representativepopulation of genome fragments, or detecting one or more members of anamplified representative population of genome fragments when bound tocapture probes on an array. Exemplary separation and detection methodsfor nucleic acids having attached binding moieties are set forth belowin further detail.

A binding moiety, detection moiety or any other useful moiety can beattached to a nucleic acid such as an amplified genome fragment usingmethods known in the art. For example, a primer used to amplify anucleic acid can include the moiety attached to a base, ribose,phosphate, or analogous structure in a nucleic acid or analog thereof.In particular embodiments, a moiety can be incorporated using modifiednucleosides that are added to a growing nucleotide strand, for example,during amplification or detection steps. Nucleosides can be modified,for example, at the base or the ribose, or analogous structures in anucleic acid analog. Thus, a method of the invention can include a stepof labeling genome fragments to produce an amplified representativepopulation of genome fragments having one or more of the modificationsset forth above. A nucleic acid primer used to amplify a gDNA in amethod of the invention can include a complementary sequence that is anylength capable of binding to a template gDNA with sufficient stabilityand specificity to prime polymerase replication activity. Thecomplementary sequence can include all or a portion of a primer used foramplification. The length of the complementary sequence of a primer usedfor amplification in a method of the invention will generally beinversely proportional to the distance between priming sites on a gDNAtemplate. Thus, amplification can be carried out with primers havingrelatively short complementary sequences including, for example, at most5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 nucleotides in length.

Those skilled in the art will recognize that specificity ofhybridization is generally increased as the length of the nucleic acidprimer is increased. Thus, a longer nucleic acid primer can be used, forexample, to increase specificity or reproducibility of replication, ifdesired. Accordingly, a nucleic acid used in a method of the inventioncan be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or morenucleotides long. Those skilled in the art will recognize that a nucleicacid probe used in the invention can also have any of the exemplarylengths set forth above.

Two general approaches to whole genome amplification that can be used inthe invention include the use of some form of randomly-primedamplification or creation of a genomic representation amplifiable byuniversal PCR. Exemplary techniques for randomly-primed amplificationinclude, without limitation, those based upon PCR, such as PEP-PCR orDOP-PCR or those based upon strand-displacement amplification such asrandom-primer amplification. An exemplary method of creating genomicrepresentations amplifiable by universal PCR is described, for example,in Lucito et al., Proc. Nat'l, Acad. Sci. USA 95:4487-4492 (1998). Oneimplementation of genomic representations is to create short genomicinserts (for example, 30-2000 bases) via restriction digestion of gDNA,and add universal PCR tails by adapter ligation. Typically,amplification or detection of gDNA is carried out with a population ofnucleic acids that hybridizes to different portions of a gDNA template.A population of nucleic acids used in the invention can include membershaving a random or semi-random complement of sequences. Thus, apopulation of nucleic acids can have members with a fixed sequencelength in which one or more positions along the sequence are randomizedwithin the population. By way of example, a population of 12mer primerscan have a sequence that is identical except at one particular position,say position 5, where any of the four native DNA nucleotides areincorporated, thereby producing a population having four differentprimer members. In a particular embodiment, multiple positions along thesequence can be combinatorially randomized. For example, a nucleic acidprimer can have 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,100 or more positions that are randomized. For example a 12mer primerthat is randomized at each position with 4 possible native DNAnucleotides will contain up to 4¹²=1.7×10⁷ members.

In particular embodiments, a population of nucleic acids used in theinvention can include members with sequences that are designed based onrational algorithms or processes. Similarly, a population of nucleicacids can include members each having at least a portion of theirsequence designed based on rational algorithms or processes. Rationaldesign algorithms or processes can be used to direct synthesis of anucleic acid product having a discrete sequence or to direct synthesisof a nucleic acid mixture that is biased to preferentially containparticular sequences.

Using rational design methods, sequences for nucleic acids in apopulation can be selected, for example, based on known sequences in thegDNA to be amplified or detected. The sequences can be selected suchthat the population preferentially includes sequences that hybridize togDNA with a desired coverage. For example, a population of primers canbe designed to preferentially include members that hybridize to aparticular chromosome or portion of a gDNA such as coding regions ornon-coding regions. Other properties of a population of nucleic acidscan also be selected to achieve preferential hybridization at positionsalong a gDNA sequence that are at a desired average, minimum or maximumlength from each other. For example, primer length can be selected tohybridize and prime at least about every 64, 256, 1000, 4000, 16000 ormore bases from each other along a gDNA sequence.

Nucleic acids useful in the invention can also be designed topreferentially omit or reduce sequences that hybridize to particularsequences in a gDNA to be amplified or detected such as known repeats orrepetitive elements including, for example, Alu repeats. Accordingly, asingle probe or primer such as one used in arbitrary-primeramplification can be designed to include or exclude a particularsequence. Similarly a. population of probes or primers, such as apopulation of primers used for random primer amplification, can besynthesized to preferentially exclude or include particular sequencessuch as Mu repeats. A population of random primers can also besynthesized to preferentially include a higher content of G and/or Cnucleotides compared to A and T nucleotides. The resulting random primerpopulation will be GC rich and therefore have a higher probability ofhybridizing to high GC regions of a genome such as gene coding regionsof a human genome which typically have a higher GC content thannon-coding gDNA regions. Conversely, Al′ rich primers can be synthesizedto preferentially amplify or anneal to AT rich regions such asnon-coding regions of a human genome. Other parameters that can be usedto influence nucleic acid design include, for example, preferentialremoval of sequences that render primers self-complementary, prone toformation of primer (timers or prone to hairpin formation orpreferential selection of sequences that have a desired maximum, minimumor average T_(m). Exemplary methods and algorithms that can be used inthe invention for designing probes include those described in US2003/0096986A1.

Primers in a population of random primers can have a region of identicalsequence such as a universal tail. A universal tail can include auniversal priming site for a subsequent amplification step or a sitethat anneals to a particular binding agent useful for isolating ordetecting amplified sequences. Methods for making and using a populationof random primers with universal tails are described, for example, inSinger et al., Nucl. Acid. Res, 25:781-786 (1997) or Grothues et al.,Nucl. Acids Res. 21:1321-2 (1993).

Those skilled in the art will recognize that any of a variety of nucleicacids used in the invention such as probes can have one or more of theproperties, or can be produced, as set forth above including in theexamples provided with respect to (primers.

A method of the invention for amplifying a genome can include a step ofcontacting a gDNA with a polymerase under conditions forrepresentationally amplifying the genomic DNA. The type of polymeraseand conditions used for amplification in a method of the invention canbe chosen to obtain genome fragments having a desired length, Inparticular embodiments, relatively small fragments can be obtained in amethod of the invention, for example, by amplifying gDNA with a.polymerase of low processivity or by fragmenting a gDNA template or itsamplification products with a nucleic acid cleaving agent such as anendonuclease or chemical agent. For example, a method of the inventioncan be used to obtain an amplified representative population of genomefragments that are, without limitation, at most about 10 kb, 5 kb, 4 kb,3 kb, 2 kb, 1 kb, 0.8 kb, 0.6 kb, 0.5 kb, 0.4 kb, 0.2 kb, or 0.1 kb inlength.

In alternative embodiments, a method of the invention can be used toamplify gDNA to form relatively large genomic DNA fragments. Inaccordance with such embodiments, a method of the invention can be usedto obtain an amplified representative population of genome fragmentsthat are at least about 10 kb, 15 kb, 20 kb, 25 kb, 30 kb or more inlength.

An amplified representative population including genome fragments havingrelatively small size can be obtained, for example, by amplifying thegDNA with a polymerase of low processivity. A low processivitypolymerase used in a method of the invention can synthesize less than100 bases per polymerization event. Shorter fragments can be obtained ifdesired by using a polymerase that synthesizes less than 50, 40, 30, 20,10 or 5 bases per polymerization event under the conditions ofamplification. A non-limiting advantage of using a low processivitypolymerase for amplification is that relatively small fragments areobtained, thereby allowing efficient hybridization to nucleic acidarrays. A low-processivity polymerase can be particularly useful foramplifying a fragmented genome sample, As set forth below, particularlyuseful methods of individual analysis can include, for example, captureof fragments at discrete locations in an array of probes.

In a particular embodiment, a denatured or single-stranded genomic DNAtemplate can be amplified using a low processivity polymerase in amethod of the invention. A gDNA template can be denatured, for example,by heat, enzymes such as helicase, chemical agents such as salt ordetergents, pH or the like. Exemplary polymerases that are capable oflow processivity and useful for amplifying gDNA in the inventioninclude, without limitation, Taq polymerase, T4 polymerase, “monomeric”E. coli Pol III (lacking the beta subunit), or E. coli DNA Poll or its5′ nuclease deficient fragment known as Klenow polymerase.

The invention further provides embodiments in which amplification occursunder conditions where the gDNA template is not denatured. An exemplarycondition is a temperature at which an isolated genomic DNA remainssubstantially double stranded. Conditions in which high temperaturedenaturation of DNA is not required are typically referred to asisothermal conditions. Genomic DNA can be amplified under isothermalconditions in the invention using a polymerase having strand displacingactivity. In particular embodiments, a polymerase having both lowprocessivity and strand displacing activity can be used to obtain anamplified representative population of genome fragments. Exemplarypolymerases that are capable of low processivity and strand displacementinclude, without limitation, E. coli Pol I, exo⁻ Klenow polymerase orsequencing grade T7 exo-polymerase. Generally, polymerase activity,including, for example, processivity and strand displacement activity,can be influenced by factors such as pH, temperature, ionic strength,and buffer composition. Those skilled in the art will know which typesof polymerases and conditions can be used to obtain fragments having adesired length in view of that which is known regarding the activity ofthe polymerases as described, for example, in Eun, Enzymology Primer forRecombinant DNA Technology, Academic Press, San Diego (1996) or will beable to determine appropriate polymerases and conditions by systematictesting using known assays, such as gel electrophoresis or massspectrometry, to measure the length of amplified fragments.

E. coli Pol for its Klenow fragment can be used for isothermalamplification of a genome to produce small genomic DNA fragments, forexample, in a low salt (1=0.085) reaction incubated at a temperaturebetween about 5° C. and 37° C. Exemplary buffers and pH conditions thatcan be used to amplify gDNA with Klenow fragment include, for example,50 mM Tris HCl (pH 7.5), 5 mM MgCl₂, 50 mM NaCl, 50 ug/ml bovine serumalbumin (BSA), 0.2 mM of each dNTP, 2 ug (microgram) random primer(n=6), 10 ng gDNA template and 5 units of Klenow exo-incubated at 37° C.for 16 hours. Similar reaction conditions can be run where one or morereaction component is omitted or substituted. For example, the buffercan be replaced with 50 mM phosphate (pH 7.4) or other pH values in therange of about 7.0 to 7.8 can be used. A gDNA template to be amplifiedcan be provided in any of a variety of amounts including, withoutlimitation, those set forth previously herein. In an alternativeembodiment, conditions for amplification can include, for example, 10 nggenomic DNA template, 2 mM dNTPs, 10 mM MgCl₂. 0.5 U/ul (microliter)polymerase, 50 uM (micromolar) random primer (n=6) and isothermalincubation at 37° C. for 16 hours.

In particular embodiments, an amplification reaction can be carried outin two steps including, for example, an initial annealing step followedby an extension step. For example, 10 ng gDNA can be annealed with 100uM random primer (n=6) in 30 ul of 10 mM Tris-Cl (pH 7.5) by briefincubation at 95° C. The reaction can be cooled to room temperature andan annealing step carried out by adding an equal volume of 20 mM Tris-Cl(pH 7.5), 20 mM MgCl₂, 15 mM dithiothreitol, 4 mM dNTPs and 1 U/ulKlenow exo- and incubating at 37° C. for 16 hrs. Although exemplifiedfor Klenow-based amplification, those skilled in the art will recognizethat separate annealing and extension steps can be used foramplification reactions carried out with other polymerases such as thoseset forth below.

In particular embodiments, primers having random annealing regions ofdifferent lengths (n) can be substituted in the Klenow-basedamplification methods. For example, the n=6 random primers in the aboveexemplary conditions can be replaced with primers having other randomsequence lengths including, without limitation, n=7, 8, 9, 10, 11 or 12nucleotides. Again, although exemplified for Klenow-based amplification,those skilled in the art will recognize that random primers havingdifferent random sequence lengths (n) can be used for amplificationreactions carried out with other polymerases such as those set forthbelow.

T4 DNA polymerase can be used for amplification of single stranded ordenatured gDNA, for example, in 50 mM HEPES pH 7.5, 50 mM Tris-HCl pH8.6, or 50 mM glycinate pH 9.7. A typical reaction mixture can alsocontain 50 mM KCl, 5 mM MgCl₂, 5 mM dithiothreitol (DTT), 40 ug/ml gDNA,0.2 mM of each dNTP, 50 ug/ml BSA, 100 uM random primer (n=6) and 10units of T4 polymerase incubated at 37° C. for at least one hour.Temperature cycling can be used to displace replicate strands formultiple rounds of amplification.

T7 polymerase is typically highly processive allowing polymerization ofthousands of nucleotides before dissociating from a template DNA.Typical reaction conditions under which T7 polymerase is highlyprocessive are 40 mM Tris-HCl pH 7.5, 15 mM MgCl₂, 25 mM NaCl, 5 mM DTT,0.25 mM of each dNTP, 50 ug/ml single stranded gDNA, 100 uM randomprimer (n=6) and 0.5 to 1 unit of T7 polymerase. However, attemperatures below 37° C. processivity of T7 polymerase is greatlyreduced. Processivity of T7 polymerase can also be reduced at high ionicstrengths, for example above 100 mM NaCl. Form II T7 polymerase is nottypically capable of amplifying double stranded DNA. However, Form I T7polymerase and modified T7 polymerase (SEQUENASE™ version 2.0 whichlacks the 28 amino acid region Lys118 to Arg 145) can catalyze stranddisplacement replication. Accordingly, small genome fragments can beamplified in a method of the invention using a modified T7 polymerase ormodified conditions such as those set forth above. In particularembodiments, SEQUENASE™ can be used in the presence of E. coli singlestranded binding protein (SSB) for increased strand displacement. SSBcan also be used to increase processivity of SEQUENASE™, if desired.

Taq polymerase is highly processive at temperatures around 70° C. whenreacted with a 10 fold molar excess of template and random primer (n=6).An amplification reaction run under these conditions can further includea buffer such as Tris-HCl at about 20 mM, pH of about 7, about 1 to 2 mMMgCl₂, and 0.2 mM of each dNTP. Additionally a stabilizing agent can beadded such as glycerol, gelatin, BSA or a non-ionic detergent. Taqpolymerase has low processivity at temperatures below 70° C.Accordingly, small fragments of gDNA can be obtained by using Taqpolymerase at a low temperature in a method of the invention, or inanother condition in which Taq has low processivity. In anotherembodiment, the Stoffel Fragment, which lacks the N-terminal 289 aminoacid residues of Taq polymerase and has low processivity at 70° C., canbe used to generate relatively small gDNA fragments in a method of theinvention. Taq can be used to amplify single stranded or denatured DNAtemplates in a method of the invention. Temperature cycling can be usedto displace replicate strands for multiple rounds of amplification.

Those skilled in the art will recognize that the conditions foramplification with the various polymerases as set forth above areexemplary. Thus, minor changes that do not substantially alter activitycan be made. Furthermore, the conditions can be substantively changed toachieve a desired amplification activity or to suit a particularapplication of the invention.

The invention can also be carried out with variants of theabove-described polymerases, so long as they retain polymerase activity.Exemplary variants include, without limitation, those that havedecreased exonuclease activity, increased fidelity, increased stabilityor increased affinity for nucleoside analogs. Exemplary variants as wellas other polymerases that are useful in a method of the inventioninclude, without limitation, bacteriophage phi29 DNA polymerase (U.S.Pat. Nos. 5,198,543 and 5,001,050), exo(−)Bca DNA polymerase (Walker andLinn. Clinical Chemistry 42:1604-1608 (1996)), phage M2 DNA polymerase(Matsumoto et al., Gene 84:247 (1989)), phage phiPRD 1 DNA polymerase(Jung et al., Proc. Natl. Acad. Sci. USA 84:8287 (1987)), exo(−)VENT™DNA polymerase (Kong et al., J Biol. Chem. 268.1965-1975 (1993)), T5 DNApolymerase (Chatterjee et al., Gene 97:13-19 (1991)), and PRD1 DNApolymerase (Zhu et al., Biochim. Biophys. Acta. 1219:267-276 (1994)).

A further polymerase variant that is useful in a method of the inventionis a modified polymerase that, when compared to its wild type unmodifiedversion, has a reduced or eliminated ability to add non-templatedirected nucleotides to the 3′ end of a nucleic acid. Exemplary variantsinclude those that affect activity of the polymerase toward adding alltypes of nucleotides or one or more types of nucleotides such aspyrimidine nucleotides, purine nucleotides, A, C, T, U or G.Modifications can include chemical modification of amino acid groups inthe polymerase or sequence mutations such as deletions, additions orreplacements of amino acids. Examples of modified polymerases havingreduced or eliminated ability to add non-template directed nucleotidesto the 3′ end of a nucleic acid are described, for example, in U.S. Pat.No. 6,306,588 or Yang et al., Nucl. Acids Res. 30:4314-4320 (2002). In aparticular embodiment, such a polymerase variant can be used in an SBEor ASPE detection method described herein.

In particular embodiments of the invention, a double stranded genomicDNA that is to be amplified by a strand displacing polymerase can bereacted with a nicking agent to produce single strand breaks in thecovalent structure of the genomic DNA template. The introduction ofsingle strand breaks in a gDNA template can be used, for example, toimprove amplification efficiency or reproducibility in isothermalamplification. Nicking can be used, for example, in a random primeramplification reaction or arbitrary-primed amplification reaction. Anon-limiting advantage of introducing single-strand breaks in anamplification reaction is that it can be used in place of heatdenaturation. Heat denaturation is deleterious to certain random-primedamplification reactions as described, for example, in Lage et al.,Genome Res. 13:294-307 (2003). In this regard, locations at which a gDNAtemplate is nicked can provide priming sites for polymerase activity.Thus, contacting a gDNA with a nicking agent can increase the number ofpriming sites in the gDNA template, thereby improving amplificationefficiency. The number of nicks or location of nicks or both can beinfluenced by use of particular conditions that favor a desired nickingactivity level or use of a nicking agent that is sequence specific.Thus, use of a nicking agent can improve the reproducibility ofamplification.

Accordingly, the invention further provides a method of amplifyinggenomic DNA that includes the steps of: (a) providing isolated doublestranded genomic DNA; (b) contacting the double stranded genomic DNAwith a nicking agent, thereby producing nicked double stranded genomicDNA; and (c) contacting the nicked double stranded genomic DNA with astrand displacing polymerase and a plurality of primers, wherein thegenomic DNA is amplified. As set forth above, the plurality of primerscan be a population of random primers, for example, in a random primeramplification reaction.

A nicking agent used in a method of the invention can be any physical,chemical, or biochemical entity that cleaves a covalent bond connectingadjacent sequences in a first nucleic acid strand producing a product inwhich the adjacent sequences are hybridized to the same complementarystrand. Exemplary nicking agents include, without limitation, singlestrand-nicking enzymes such as DNAse I, N.BstNBI, MutH, or genellprotein of bacteriophage; chemical reagents such as free radicals; orultrasound.

A nicking agent can be contacted with a double stranded gDNA by mixingthe agent and gDNA together in solution. Those skilled in the art willknow or be able to determine appropriate conditions for nicking the gDNAbased on that which is known in the art regarding activity of thenicking agent as available, for example, from various commercialsuppliers such as Promega Corp. (Madison, Wis.), or Roche AppliedSciences (Indianapolis, Ind.). A chemical or biological nicking agentcan be one that is exogenous to the genomic DNA, having come from asource that is different from the DNA. Alternatively, a nicking agentthat is normally found with the genomic DNA in its native environmentcan be contacted with the gDNA in a method of the invention.

Such an endogenous nicking agent can be activated to increase itsnicking activity or it can be isolated from the genomic DNA andsubsequently mixed with the gDNA, for example, at a higher concentrationcompared to its native environment with the gDNA. A nicking agent,whether endogenous or exogenous to a gDNA, can be isolated prior tobeing contacted with the gDNA in a method of the invention.

Those skilled in the art will understand that an amplifiedrepresentative population of genome fragments can be provided from afreshly isolated sample or one that has been stored under appropriateconditions for preserving the integrity of the sample. Thus, a sampleprovided in a method of the invention can include agents that stabilizethe fragments, so long as the agents do not interfere with hybridizationand detection steps and other steps used in the various embodiments setforth herein. In cases where a stabilizing agent that interferes withthe methods is included in a sample, the fragments can be separated fromthe agent using known purification and separation methods, Those skilledin the art will know or be able to readily determine appropriateconditions for storing a representative population of genome fragmentsbased on conditions known in the art for storing nucleic acids asdescribed, for example, in Sambrook et al., supra, (2001) and in Ausubelet al., supra, (1998). In particular embodiments, a gDNA can beamplified by a method that utilizes random or degenerate oligonucleotideprimed polymerase chain reaction (PCR) with heat denatured gDNAtemplates. An exemplary method is known as primer extensionpreamplification (PEP). This technique uses random 15-mers incombination with Taq DNA polymerase to initiate copies throughout thegenome. This technique can be used to amplify genomic DNA from as littleas a single cell using, for example, conditions described in Zhang etal., Proc. Natl. Acad. Sci, USA, 89:5847-51 (1992); Snabes et al., Proc.Natl. Acad., Sci. USA, 91:6181-85 (1994,); or Barrett et al., NucleicAcids Res., 23:3488-92 (1995).

Another gDNA amplification method that is useful in the invention isTagged PCR which uses a population of two-domain primers having aconstant 5′ region followed by a random 3′ region as described, forexample, in Grothues et al. Nucleic Acids Res. 21(5):1321-2 (1993). Thefirst rounds of amplification are carried out to allow a multitude ofinitiations on heat denatured. DNA based on individual hybridizationfrom the randomly-synthesized 3′ region. Due to the nature of the 3′region, the sites of initiation will be random throughout the genome.Thereafter, the unbound primers can be removed and further replicationcan take place using primers complementary to the constant 5′ region.

A further approach that can be used to amplify gDNA in a method of theinvention is degenerate oligonucleotide primed polymerase chain reaction(DOP-PCR) under conditions described, for example, by Cheung et al.,Proc. Natl. Acad. Sci. USA, 93:14676-79 (1996) or U.S. Pat. No.5,043,272. Low amounts of gDNA, for example, 15 pg of human gDNA, can beamplified to levels that are conveniently detected in the methods of theinvention. Reaction conditions used in the methods of Cheung et al, canbe selected for production of an amplified representative population ofgenome fragments having near complete coverage of the human genome.Furthermore modified versions of DOP-PCR, such as those described byKittler et al. in a protocol known as LL-DOP-PCR (Long products from LowDNA quantities-DOP-PCR) can be used to amplify gDNA in accordance withthe invention (Kittler et al., Anal. Biochem. 300:237-44 (2002)).

Primer-extension preamplification polymerase chain reaction (PEP-PCR)can also be used in a method of the invention in order to amplify gDNA.Useful conditions for amplification of gDNA. using PEP-PCR include, forexample, those described in Casas et al., Biotechniques 20:219-25(1996).

Amplification of gDNA in a method of the invention can also be carriedout on a gDNA template that has not been denatured. Accordingly, theinvention can include a step of producing an amplified representativepopulation of genome fragments from a gDNA template under isothermalconditions. Exemplary isothermal amplification methods that can be usedin a method of the invention include, but are not limited to, MultipleDisplacement Amplification (MDA) under conditions such as thosedescribed in Dean et al., Proc Natl. Acad. Sci USA 99:5261-66 (2002) orisothermal strand displacement nucleic acid amplification as describedin U.S. Pat. No. 6,214,587. Other non-PCR-based methods that can be usedin the invention include, for example, strand displacement amplification(SDA) which is described in Walker et al., Molecular Methods for VirusDetection, Academic Press, Inc., 1995; U.S. Pat. Nos. 5,455,166, and5,130,238, and Walker et al., Nucl, Acids Res. 20:1691-96 (1992) orhyperbranched strand displacement amplification which is described inLage et al., Genome Research 13:294-307 (2003). Isothermal amplificationmethods can be used with the strand-displacing .PHI.29 polymerase or BstDNA polymerase large fragment, 5T-→3′ exo⁻ for random primeramplification of genomic DNA. The use of these polymerases takesadvantage of their high processivity and strand displacing activity.High processivity allows the polymerases to produce fragments that are10-20 kb in length. As set forth above, smaller fragments can beproduced under isothermal conditions using polymerases having lowprocessivity and strand-displacing activity such as Klenow polymerase.

In particular embodiments of the invention, a genomic DNA or populationof amplified gDNA fragments can be in vitro transcribed into genomic RNA(gRNA) fragments, Creation of gRNA in a method of the invention offersseveral non-limiting advantages for detection of typable loci in primerextension assays such as DNA array-based primer extension assays,Array-based primer extension typically includes a step of hybridizing atarget DNA to an immobilized probe DNA and subsequent modification orextension of the probe-target hybrid with a DNA polymerase. These assayscan often be compromised by artifacts arising from unwanted formation ofprobe-probe hybrids, due to their physical proximity on the arraysurface, and subsequent ectopic extension of these probe-probe hybrids.In embodiments of the invention where gDNA is converted into gRNA, suchartifacts can be avoided because DNA polymerase is replaced with reversetranscriptase (RT) which does not efficiently modify or extendprobe-probe hybrids because they are DNA-DNA hybrids and reversetranscriptase is selective for hybrids having an RNA template.Furthermore, the use of gRNA and reverse transcriptase for detection oftarget probe hybrids minimizes ectopic extension in a directhybridization array-based primer extension assay. In an array-basedprimer extension reaction both inter-probe and intra-probeself-extension (ectopic extension) can lead to high-backgrounds. Use ofRT and gRNA prevent artifacts due to ectopic extension because, althoughRT can easily extend a DNA probe hybridized to an RNA target, it willnot efficiently extend DNA-DNA complexes.

Accordingly, the invention provides a method for detecting typable lociof a genome. The method includes the steps of (a) in vitro transcribinga population of amplified gDNA fragments, thereby obtaining genomic RNA(gRNA) fragments; (b) hybridizing the gRNA fragments with a plurality ofnucleic acid probes having sequences corresponding to the typable loci;and (c) detecting typable loci of the gRNA fragments that hybridize tothe probes.

A diagrammatic example of a method for amplifying gDNA to produce gRNAfragments is shown in FIG. 8 , As shown in Panel 8A, gDNA can beamplified with DNA polymerase and a population of random DNA primers toproduce a representative population of genome fragments prior to an invitro transcription step. In the example shown, gDNA is Random-primedlabeled (RPL) using a population of primers including a random region of9 nucleotides and a fixed region having a universal priming sequence(U1) and a T7 promoter sequence (T7). In the example shown in FIG. 8 ,the random sequence is 9 nucleotides long. However, it will beunderstood that any of a variety of random sequence lengths can be usedto suit a particular application of the invention including, forexample, a random sequence that is 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14,15 or more nucleotides long. Furthermore, a random sequence of a primerused in a method of the invention can include interspersed positionshaving a fixed nucleotide or regions having a fixed sequence of two ormore nucleotides, if desired.

As shown in Panel B, the representative population of T7 promoterlabeled genome fragments can be in vitro transcribed to gRNA form usinga T7 RNA polymerase and a complementary T7 primer (cT7). Transcriptionof gDNA to gRNA fragments can also be carried out with other promoterssuch as T3 or SP6 and their respective polymerases as set forth infurther detail below.

A gRNA-based representative population of genome fragments produced byin vitro transcription can be manipulated and detected in any of avariety of ways as set forth herein. For example, the gRNA-based genomefragments produced by the methods exemplified in FIG. 8B will have U1labeled tails. These tails can be used, for example, to isolate the gRNAfragments from gDNA and other amplification reaction components using acomplementary capture sequence attached to a solid phase. Genomic RNAfragments can be detected or copied into DNA using a reversetranscriptase. The gRNA-based representative population of genomefragments can be detected directly using methods such as those set forthbelow or, alternatively, can be copied into DNA prior to detection. Asshown in the exemplary amplification step of FIG. 8C, the population ofgRNA fragments can be replicated using locus-specific primers,optionally having a second universal sequence (U2), and a reversetranscriptase. This step can be followed by amplification usinguniversal PCR with U1 and U2 primers Thus, the gRNA. fragments can bereplicated to produce a locus-specific, amplified representativepopulation of genome fragments. As set forth below in further detail,reverse transcriptase-directed replication of the gRNA with locusspecific primers can provide complexity reduction and, if desired, canadd a U2 universal priming site. In embodiments where the U2 sequence ispresent, the population of genome fragments produced by replication withlocus specific primers will each have flanking U1 and U2 sequences thatare useful for detecting or amplifying the population. Thus, the fullyextended products can be amplified in a universal PCR reaction primed atthe U1 and U2 primer sites.

Moreover, as shown in FIG. 8D, a “primer-dimer” cannot be extended inthe detection step because reverse transcriptase cannot extend a DNAtemplate very efficiently. In contrast, a DNA polymerase can extend theL1-L2 primer dimer potentially leading to detection artifacts. Thus, theuse of gRNA-based representative populations of genome fragments canprovide the non-limiting advantage of avoiding artifacts in somemultiplex detection methods. Thus, the use of gRNA can provide theadvantage of increased efficiency for multiplexed detection of largenumbers of typable loci.

A nucleic acid primer used in a method of the invention to transcribegDNA into a gRNA-based representative population of genome fragments orto reverse transcribe gRNA can have length, composition or otherproperties as set forth herein in regard to primers used with otherpolymerases and templates. Those skilled in the art will know or be ableto determine appropriate properties of a nucleic acid primer for use inan in vitro transcription or reverse transcriptase step of the inventionbased on the guidance and teaching set forth herein and that which isknown regarding reverse transcriptases or RNA polymerases as set forthbelow and described, for example, in Fun et al., supra (1996).

Furthermore, although the primer populations exemplified above in regardto the embodiment of FIG. 8 have a single U1 sequence and a single U2sequence, it will be understood that a population of primers useful inthe invention can include more than one constant sequence region. Thus,a plurality of random primer sub-populations, each having differentconstant sequence regions, can be present in a larger population usedfor hybridization or amplification in a method of the invention. Any RNApolymerase that is capable of synthesizing a complementary RNA from aDNA template can be used in a method of the invention. An exemplary RNApolymerase useful in the invention is T7 RNA polymerase. Conditions thatcan be used in a method of the invention for in vitro transcription withT7 RNA polymerase include, without limitation, 40 mM Tris-HCl pH 8.0(37° C.), 6 mM MgCl₂, 5 mM DTT, 1 mM spermidine, 50 ug/ml BSA, 40 ug/mlgDNA fragments including a phage promoter, 0.5 to 8.5 mM NTPs, and 200to 300 units T7 RNA polymerase in 50 microliters. Another RNA polymerasethat can be used in a method of the invention is SP6 RNA polymerase.Exemplary conditions for use include, without limitation, 40 mM Tris-HClpH 8.0 (25° C.), 6 mM MgCl₂, 10 mM DTT, 2 mM spermidine, 50 ug/ml BSA,50 ug/ml gDNA fragments containing an SP6 promoter, 0.5 mM of each NTP,and 10 units SP6 RNA polymerase in 50 microliters.

T3 RNA polymerase can also be used in a method of the invention for invitro transcription, for example, under conditions including 50 mMTris-HCl pH 7.8 (37° C.), 25 mM NaCl, 8 mM MgCl₂, 5 mM DTT, 2 mMspermidine, 50 ug/ml BSA, 50 ug/ml gDNA fragments containing a T3promoter, 0.5 mM of each NTP, and T3 RNA polymerase in 50 microliters.

Any reverse transcriptase (RT) that catalyzes the synthesis ofcomplementary DNA from an RNA template can be used in a method of theinvention. Exemplary RTs that can be used in a method of the inventioninclude, but are not limited to, those from retroviruses such as avianmyoblastosis virus (AMV) RT, Moloney murine leukemia virus (MoLV) RT,RT, or Rouse sarcoma virus (RSV) RT. Generally, a reverse transcriptionreaction used in a method of the invention will include an RNA template,one or more dNTPs and a nucleic acid primer with a 3′ OH group. RNAseinhibitors can be added, if desired, to inhibit degradation of thetranscribed product. Particular reaction conditions can be used to suita particular RT or a particular application of the invention.

Useful conditions for modification or elongation with AMV RT include,for example, 50 mM Tris-HCl (pH 8.3 at 42° C.), 150 mM NaCl (or 100 mMKCl), 6 to 10 mM MgCl₂, 1 mM DTT, 50 ug/ml BSA, 50 units RNasin, 0.5 mMSpermidine HCL, 4 mM NA-PP_(i), 0.2 mM of each dNTP, 1-5 ug gRNA, 0.5 to2.5 ug primer and 10 units AMV RT in 50 microliters. However it is alsopossible to perform the reaction at pH 8.1 at 25° C. with otherwisesimilar conditions. Other conditions that can be used for AMV RTactivity and in particular to inhibit DNA-dependent DNA synthesis aredescribed, for example, in Lokhava et al., FEBS Lett. 274: 156-158(1990) or Lokhava et al., Mol. Biol. (USSR) 24:396-407 (1990).

In embodiments where MoLV RT is used, exemplary conditions formodification or elongation include, without limitation, 50 mM Tris-HCl(pH 8.1 at 25° C.), 75 mM KCl, 3 mM MgCl₂, 10 mM DTT, 100 ug/ml BSA, 20units RNasin, 50 ug/ml actinomycin D, 0.5 mM of each dNTP, 5-10 ug gRNA,0.5 to 4 ug primer and 200 units MoLV RT in 50 microliters.

An RT used in a method of the invention can also be from anon-retroviral source including, for example, DNA viruses such ashepatitis B virus or caulimovirus, bacteria such as Myxococcus xanthusor some strains of E. coli, yeast such as those bearing the Tyretrotransposon, fungi, invertebrates such as those bearing thecopia-like element of Drosophila, or plants. Furthermore, if desiredreverse transcription can be carried out in a method of the inventionusing a DNA polymerase that has RT activity such as E. coli DNA Pol I.However, for the reasons set forth above, it may be desired to carry outreverse transcription under conditions in which activity toward DNAtemplates is inhibited or substantially absent, for example, using an RTthat is not capable of DNA-dependent DNA synthesis or using conditionssuch as a pH, ionic strength or Mg²⁺ concentration that inhibitDNA-dependent DNA synthesis. Furthermore, an inhibitor of DNA-dependentDNA synthesis such as actinomycin D or pyrophosphate (Na-PP_(i)) can beadded if desired.

An exemplary DNA polymerase that is capable of RT activity is Tth polwhen used in the presence of Mn²⁺. Exemplary conditions for reversetranscription of gRNA with Tth pol RT include, without limitation, 50 mMTris-Cl (pH 8.8), 16 mM NH₄SO₄, 1 mM MnCl₂, 200 μM dNTPs, 0.25 U/μl Tthpol, 100 fmol/μl RNA template at 70° C. for 20 min.

Amplification of gDNA in a method of the invention can be carried outsuch that an amplified representative population of gnome fragmentshaving a desired complexity is produced. For example, an amplifiedrepresentative population of genome fragments having a desiredcomplexity can be produced by specifying the frequency or diversity ofpriming or fragmentation events that occur during an amplificationreaction. Accordingly, the invention can be used to produce an amplifiedrepresentative population of genome fragments having high or lowcomplexity depending upon the desired use of the population offragments. Several of the amplification conditions set forth above andin the Examples below provide high complexity representations. A methodof the invention can include a complexity reduction step or can becarried out with an amplification method that produces a low complexityrepresentation, if desired.

An exemplary method for producing a low complexity representation islinker adaptor-PCR which calls for an initial random digestion of DNAwith a restriction endonuclease, ligation of the digested fragments toan adaptor oligonucleotide and PCR amplification of heat denaturedadaptor derivatized fragments as described, for example, in Lucito etal., Genome Res. 10:1726-36 (2000). Altering the conditions of gDNAdigestion in the method can be used to influence the complexity of theamplified representative population of genome fragments that isproduced. In particular, a low complexity representation can be obtainedusing an infrequent-cutting endonuclease having, for example, a 6 baseor longer recognition motif. Accordingly, a frequent cutter can be usedto obtain a high complexity representation. For example, Dpn II, whichrecognizes the four nucleotide site GATC, and thus restricts gDNArelatively frequently, can produce a representative population of humangenome fragments that that contains about 70% of the genome. Incontrast, a relatively infrequent cutter can be used to produce a lowcomplexity representation. For example, Bgl II, which recognizes the sixnucleotide site AGATCT and thus restricts gDNA relatively infrequently,can be used to produce a representative population of human genomefragments that contains only approximately 2.5% of a genome.Furthermore, a gDNA can be fragmented to an average length that issmaller than the processivity of the polymerase used for amplification,thereby reducing the complexity of the amplified representativepopulation of genome fragments that is produced.

A further method for producing a low complexity representation is theuse of two or more adaptors for anchored linker adaptor PCR. Inparticular embodiments complexity reduction can be achieved byfragmenting a gDNA sample using at least two restriction enzymes;ligating adaptors to the resulting fragments; and selectively amplifyingthe fragments that were cut on one end by one restriction enzyme and onthe other end by a different restriction enzyme. If one enzyme is a6-cutter and the other is a 4-cutter, the representation will beanchored about the 6-cutter sites with an average size determined byfrequency of the 4-cutter digestion (about every 256 bases). This is auseful size for PCR-based amplification. The complexity of the resultingsample can be regulated by choosing enzymes that cut with a particularfrequency. Selective amplification can also be accomplished by designingone adaptor to have a 5′ overhang and the second adaptor to have a 3′overhang where the overhangs have the annealing sites for amplificationprimers used to replicate the fragments. Exemplary conditions for theuse of multiple adaptors for complexity reduction are described in US2003/0096235 A1.

Complexity reduction can also be carried out in a locus-specific manner.Accordingly, the invention further provides a method of producing areduced complexity, locus-specific, amplified representative populationof genome fragments, The method includes the steps of (a) replicating anative genome with a plurality of random primers, thereby producing anamplified representative population of genome fragments; (b) replicatinga sub-population of the amplified representative population of genomefragments with a plurality of different locus-specific primers, therebyproducing a locus-specific, amplified representative population ofgenome fragments; and (c) isolating the sub-population, therebyproducing a reduced complexity, locus-specific, amplified representativepopulation of genome fragments,

An exemplary method that can be used for complexity reduction isamplification to produce gRNA fragments as shown in FIG. 8 and describedabove. A diagrammatic example of a method for producing a reducedcomplexity, locus-specific, amplified representative population ofgenome fragments is shown in FIG. 9 . As shown in FIG. 9A a gDNA samplecan be amplified by a Random-primed labeling (RPL) technique employing apopulation of nucleic acid primers each having a random 3′ sequence forannealing to the gDNA and a 5′ universal priming tail (U1 sequence).Thus, a random-primed labeling reaction can produce an amplifiedrepresentative population of genome fragments flanked by a universalpriming site, In the example shown in FIG. 9 , the random sequence has 9nucleotides. However, it will be understood that any of a variety ofrandom sequence lengths or compositions can be used to suit a particularapplication of the invention including, for example, those set forthpreviously herein. In general, as the length of the random annealingportion of a population of random primers is reduced the number ofpotential annealing sites on a genome will be increased, therebyincreasing the complexity of the amplified representation.

As shown in FIG. 9B, an amplified representative population of genomefragments can be isolated from genomic DNA, for example, byimmobilization on solid phase beads. In the example of FIG. 9Aimmobilization of the amplified fragments can be facilitated by a biotinbound to the N₉-U1 primer. The biotinylated amplification product can becaptured by a solid phase that is derivatized with avidin orstreptavidin and, if desired, subsequently isolated from the gDNAtemplate. Other exemplary capture moieties and their immobilizedreceptors that can be used in a primer for random primer amplificationare set forth above. Thus, a method of amplifying gDNA can furtherinclude a step of capturing or isolating an amplified representativepopulation of genome fragments. Exemplary substrates that can be used tocapture or isolate an amplified representative population of genomefragments include, for example, those set forth below in regard toseparation of single stranded nucleic acids from nucleic acid hybrids.

Those skilled in the art will recognize that amplified genome fragmentscan be separated from other reaction components in a method of theinvention using a solid phase substrate as exemplified above. Similarlyamplified genome fragments can be separated based on other properties ofthe fragments such as their size. Thus, filtration or chromatographymethods such as size exclusion chromatography can be used to separategenome fragments from other reaction components such as probes that arenot ealed.

A method of the invention can include a step of replicating asub-population of the amplified representative population of genomefragments with a plurality of different locus-specific primers eachhaving a 3′ locus specific sequence region and a 5′ constant sequenceregion. Continuing with the example of FIG. 9B, the immobilized randomprimer amplified product can be hybridized with a population ofdifferent primers having different locus-specific 3′ sequencesidentified as L1, L2 or L3, and a 5′ second universal tail (U2). At thispoint a washing step can be included, if desired, to remove mis-annealedand excess primers. Conditions for washing can include any that removenon-specifically bound nucleic acids while maintaining specific hybrids.Primer extension can then be used to replicate a subpopulation of theamplified representative population of genome fragments having sequencescomplementary to the locus-specific primers. This subpopulation willhave lower complexity compared to the original gDNA and the amplifiedpopulation of genome fragments that was produced with the N₉-U1 primer.Furthermore, the complexity reduction will be locus specific due toselection with the locus-specific primers in the second amplificationstep. The number of different locus-specific primers and length of thelocus-specific sequences can be altered to increase or decrease thecomplexity of a representation obtained in a method of the invention.

Extension of the U2 containing primers along the full length of thecaptured fragments in the example shown in FIG. 913 will produce alocus-specific, amplified representative population of genome fragmentslabeled with the first constant region (U1) and the second constantregion (U2). Thus, the fully extended products can be amplified in auniversal PCR reaction primed at the U1 and U2 primer sites. Accordinglya method of the invention can include a step of replicating a reducedcomplexity, locus specific, amplified representative population ofgenome fragments with complementary primers to flanking first and secondconstant regions. Furthermore, detection of the fragments can be madebased on the presence of both U1 and U2 sequences, for example, usingtechniques described below in regard to detection of modified OLAprobes.

Complexity reduction can also be carried out by removing particularsequences from a population of genome fragments. In one embodiment, highcopy number or abundant sequences in a sample of gnome fragments can beinhibited from hybridizing to detection or capture probes. For example,Cot analysis can be used in which abundant species are kineticallydriven to reanneal while leaving the single copy species in a singlestranded state capable of hybridization to probes. Thus in particularembodiments, a sample of genome fragments can be pre-treated with cotoligonucleotides that are complementary to particular repeatedsequences, or o other sequences that are desired to be titrated out ofthe sample, prior to exposure of the sample to an array of probes. Inanother example, a sample of genome fragments can be cooled to atemperature and for short time period that are sufficient for asubstantial fraction of over-represented sequences to re-anneal butinsufficient for substantial re-annealing of sequences present in lowcopy numbers. The resulting sample will have a reduced amount ofrepeated sequences available for subsequent interaction with an array ofprobes.

Undesired fragments that form double stranded species, for example, inCot analysis or genome fragment reannealing, can be separated fromsingle stranded species based on different properties of single anddouble stranded nucleic acids. In a particular embodiment, enzymes thatpreferentially cleave double stranded DNA can be used. For example,DNAse I can cleave double-stranded DNA 100 to 500 fold faster thansingle stranded DNA under known conditions. Accordingly, undesiredfragments can be removed by treatment with Cot oligonucleotides or byfragment reannealing, and treatment with DNAse I under conditions inwhich undesired fragments preferentially form double stranded speciesand get cleaved. Furthermore, other enzymes that preferentially modify,cleave or bind to double stranded species compared to single strandedspecies can be used to separate the species in a method of the inventionsuch as sequence specific restriction endonucleases or Kamchatka crabduplex-specific endonuclease.

Arbitrary-primer PCR can also be used to amplify a genomic DNA in amethod of the invention. Arbitrary-primer PCR can be carried out byreplicating a gDNA sample with a primer under non-stringent conditionssuch that the primer arbitrarily anneals to various locations in thegDNA. Subsequent PCR steps can be carried out at higher stringency toamplify the fragments generated due to arbitrary priming in the previousstep. The length, sequence or both of an arbitrary-primer can beselected in accordance with the probability of priming at particularintervals along the gDNA. In this regard, as primer length increases,the average interval between arbitrarily primed locations will increase,assuming no change in other amplification conditions. Similarly, aprimer having a sequence complementary to or similar to a repeatedsequence will prime more often, yielding shorter intervals betweenamplified fragments than a primer that lacks sequences that are similarto repeated sequences in a genome to be amplified. Arbitrary-primeramplification can be carried out under conditions similar to thosedescribed, for example, in Bassam et al., Australas Biotechnol. 4:232-6(1994). In accordance with the invention, amplification can be carriedout under isothermal conditions using an arbitrary primer, lowstringency annealing conditions, and a strand-displacing polymerase.

Another method that can be used to amplify a genome in the invention isinter-Alu PCR. In this method, primers are designed to anneal to Alusequences which are repeated throughout the genome. PCR amplificationwith these primers will yield fragments flanked by Alu repeats. Thoseskilled in the art will recognize that similar methods can be carriedout with primers that anneal to other repeated sequences in a genome ofinterest such as transcription regulatory regions, splice sites or thelike, Furthermore, primers to repeated sequences can be used inisothermal amplification methods such as those set forth herein.

The complexity and degree of representation resulting from amplificationwith a particular set of primers can be adjusted using different primerhybridization conditions. A variety of hybridization conditions can beused in the present invention, such as high, moderate or low stringencyconditions including, but not limited to those described in Sambrook etal., supra, (2001) or in Ausubel et al., supra, (1998). Stringentconditions favor specific sequence-dependent hybridization. In general,longer sequences and increased temperatures favor specificsequence-dependent hybridization. A useful guide to the hybridization ofnucleic acids is found in Tijssen, Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes, “Overview ofprinciples of hybridization and the strategy of nucleic acid assays”(1993).

Amplification and detection steps used in the invention are generallycarried out under stringency conditions which selectively allowformation of a hybridization complex in the presence of complementarysequences. Stringency can be controlled by altering a step parameterthat is a thermodynamic variable, including, but not limited to,temperature, formamide concentration, salt concentration, chaotropicsalt concentration, pH, organic solvent concentration, or the like,These parameters can also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus, if desired, certainsteps can be performed under relatively high stringency conditions toreduce non-specific binding.

Generally, high stringency conditions include temperatures that areabout 5-10° C. lower than the thermal melting point (T_(m)) for theannealing sequences at a particular ionic strength and pH. Highstringency conditions include those that permit a first nucleic acid tobind a complementary nucleic acid that has at least about 90%complementary base pairs along its length and can include, for example,sequences that are at least about 95%, 98%, 99% or 100% complementary.Stringent conditions can further include, for example, those in whichthe salt concentration is less than about 1.0 M sodium ion (or othersalts), typically about 0.01 to 1.0 M concentration at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short annealing sequences(e.g. 10 to 50 nucleotides) and at least about 60° C. for long annealingsequences (e.g., greater than 50 nucleotides). High stringencyconditions can also be achieved with the addition of helix destabilizingagents such as formamide. High stringency conditions can include, forexample, conditions equivalent to hybridization in 50% formamide, 5×Denhart's solution, 5>SSPE, 0.2% SDS at 42° C., followed by washing in0.1×SSPE, and 0.1% SDS at 65° C. Nucleic acid hybrids can be furtherstabilized by covalent modification with one or more cross-linkingagents.

Moderately stringent conditions include those that permit a firstnucleic acid to bind a complementary nucleic acid that has at leastabout 60% complementary base pairs along its length to the first nucleicacid, Depending upon the particular conditions of moderate stringencyused, a hybrid can form between sequences that have complementarity forat least about 75%, 85% or 90% of the base pairs along the length of thehybridized region. Moderately stringent conditions include, for example,conditions equivalent to hybridization in 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 65° C.7

Low stringency hybridization includes, for example, conditionsequivalent to hybridization in 10% formamide, 5× Denhart's solution,6×SSPE, 0.2% SDS at 42° C., followed by washing in 1×SSPE, 0.2% SDS, at50′ C. Denhart's solution and SSPE are well known to those of skill inthe art as are other suitable hybridization buffers (see, for example,Sambrook et al., supra (2001) or in Ausubel et al., supra (1998)).

In embodiments of the invention where a hybrid will be modified, forexample, by a polymerase, conditions can be further chosen to suit theparticular modification reaction. For example, when the modificationinvolves replication or amplification, conditions such as those setforth above in regard to particular polymerases can be used. It will beunderstood that a modifying agent such as a polymerase can be added atany point during an amplification or detection step including, forexample, prior to, during, or after the addition of nucleic acidcomponents of the modification reaction.

The methods of the invention can be used to amplify a native genome in asingle reaction step or in a single reaction vessel to produce anamplified representative population of genome fragments having highcomplexity. The ability to use a single step or reaction vessel providesa non-limiting advantage of increasing amplification efficiency comparedto methods requiring multiple steps or reaction vessels. Furthermore, inparticular embodiments a high complexity amplified representativepopulation of genome fragments can be obtained under conditions that donot require pooling of products from multiple amplification reactions.Thus, the fragments in an amplified representative population of genomefragments can be obtained in parallel rather than sequentially invarious embodiments of the invention. However, it is possible to use themethods in embodiments where different reaction steps are carried out inseparate vessels, sequentially, or where the products of multiplereactions are pooled, for example, to suit particular applications.

Further description of exemplary methods that can be used in theinvention to amplify nucleic acids, such as native genomes or fragmentsthereof, can be found in U.S. Pat. No. 6,355,431 and include polymerasechain reaction (PCR) amplification, random primed PCR, arbitrary primedPCR, strand displacement amplification, nucleic acid sequence basedamplification and transcription mediated amplification.

Following replication of a genome or population of genome fragments,nucleic acids containing a desired modification can be separated fromunmodified nucleic acids such as unreacted primers or the template. Forexample, it can be desirable to remove unextended or unreacted primersbecause unextended primers can compete with the extended or labeledprimers in a variety of the detection methods that are used in theinvention, thereby diminishing the signal. Accordingly, a number ofdifferent techniques can be used to facilitate the removal of unextendedprimers. While the discussion below is directed to amplificationreactions for clarity, it will be understood that these techniques canalso be used to separate modified and unmodified nucleic acids in adetection step.

Separation of nucleic acids can be mediated by selective incorporationof a label including, for example, one or more of the primary orsecondary labels described previously herein. Nucleic acids having anincorporated secondary label can be separated from those lacking thelabel based, for example, on binding to a receptor having specificityfor the label. The receptor can be attached, for example, to a solidphase substrate as set forth above in regard to the embodimentexemplified in FIG. 9 . Primary labels can be used to separate nucleicacids in a sorting method such as fluorescent activated cell sorting.Similarly, nucleic acids having an incorporated secondary label can beseparated from those lacking the label in a sorting method based ondetection of a receptor that provides a primary label to the nucleicacid-receptor complex. Separation can also be accomplished usingstandard size exclusion resins such as G-50 resin, ultrafiltration suchas with Amicon or Centricon columns, or ethanol-like precipitationmethods.

A nucleic acid can be conveniently labeled in a method of the inventionby a moiety introduced during an amplification or modification reactionvia a labeled primer, labeled nucleotide precursor or both. Inparticular embodiments, one or more NTPs used to replicate a nucleicacid can include a secondary detectable label that can be used toseparate modified primers from unmodified primers lacking the label.Secondary labels find particular use in detection techniques thatinclude steps for separation of labeled and unlabeled probes, such asSBE, OLA or invasive cleavage. Particularly useful labels include, butare not limited to, one of a binding partner pair; chemically,modifiable moieties; or nuclease inhibitors.

By way of example, a secondary label can be a hapten or antigen havingaffinity for an immunoglobulin, or functional fragment thereof, attachedto a solid support. Labeled nucleic acids that are bound to theimmunoglobulin can be separated from unlabeled nucleic acids by physicalseparation of the solid support and soluble fraction. In addition,avidin/biotin systems including, for example, those utilizingstreptavidin, biotin mimetics or both, can be used to separate modifiednucleic acids from those that are unmodified. Typically the smaller oftwo binding partners is attached to a nucleic acid. However, attachmentof the larger partner can also be useful, For example, the addition ofstreptavidin to a nucleic acid increases its size and changes itsphysical properties, which can be exploited for separation. Accordingly,a streptavidin labeled nucleic acid can be separated from unlabelednucleic acids in a mixture using a technique such as size exclusionchromatography, affinity chromatography, filtration or differentialprecipitation.

In embodiments, including attachment of a binding partner to a solidsupport, the solid support can be selected, for example, from thosedescribed herein with respect to detection arrays. Particularly usefulsubstrates include, for example, magnetic beads which can be easilyintroduced to the nucleic acid sample and easily removed with a magnet.Other known affinity chromatography substrates can be used as well.Known methods can be used to attach a binding partner to a solidsupport.

Typically, a method of detecting typable loci of a genome is carried outon an amplified representative population of genome fragments obtained,for example, by a method set forth above. Alternatively, typable locican be determined for a representative population of genome fragmentsderived from a genome by a method other than an amplification method. Inone embodiment, a representative population of genome fragments can beobtained by fragmenting a native genome. Exemplary methods that can beused for fragmenting a genome are set forth below. Those skilled in theart will recognize that the fragmentation methods can be used as analternative to the amplification methods described herein or, if desiredin combination with an amplification technique.

An isolated native genome can be fragmented by any physical, chemical orbiochemical entity that creates double strand breaks in DNA. Inparticular embodiments, a native genome can be digested with anendonuclease. Endonucleases useful in the methods of the inventioninclude those that cleave at a specific recognition sequence or thosethat non-specifically cleave DNA such as DNaseI. Endonucleases areavailable in the art and can be Obtained, for example, from commercialsources such as New England BioLabs (Beverley, Mass.) or Lifetechnologies Inc. (Rockville, Md.) among others. Specific endonucleasescan be used to generate polynucleotide fragments of a particular averagesize according to the frequency with which the enzyme is expected to cuta random sequence. For example, an endonuclease having a six nucleotiderecognition sequence would be expected to produce, on average, fragmentsthat are 4096 base pairs long. Average fragment length can be estimatedby treating the DNA as a random sequence and estimating the frequency ofa recognition site in the random sequence according to the relationship4^(n)=s where n is the number of bases recognized by the endonucleaseand s is the average size of the fragments produced. Incubationconditions can also be modified, as described below, to alter theenzymatic efficiency of the endonuclease, thereby altering the averagesize of the fragments produced. Using the example of an endonucleasehaving a 6 base pair recognition site, a decrease in enzymaticefficiency can produce fragments that are on average larger than 4096base pairs long.

Non-specific endonucleases can also be used to produce genome fragmentsof a desired average size. Because the endonuclease reaction isbi-molecular, the rate of fragmentation can be manipulated by alteringconditions such as the concentrations of the endonuclease, DNA or both.Specifically, a reduction in the concentration of either endonuclease,DNA or both can be used to reduce reaction rate resulting in increasedaverage fragment sizes. Increasing concentrations of eitherendonuclease, DNA recognition sequence or both will allow for increasedefficiency, approaching maximum velocity (V_(max)) for the particularenzyme leading to reduced average fragment sizes. Similar changes inconditions can also be applied to site-specific endonucleases becausetheir reactions with DNA are also bi-molecular. Other reactionconditions can also affect the rate of cleavage including, for example,temperature, salt concentration and time of reaction. Methods foraltering nuclease reaction rates to produce polynucleotide fragments ofdetermined average size are described, for example, in Sambrook et al.,supra. (2001) or in Ausubel et al., supra, (1998).

Other methods that can be used to produce genome fragments include, forexample, treatment with chemical agents that disrupt the phosphodiesterbackbone of DNA such as those that cleave bonds by a free radicalmechanism, UV light, mechanical disruption or the like. These and themethods set forth above can be used to produce genome fragments from anative genome, further cleave genome fragments, or cleave other nucleicacids used in the invention. Further exemplary mechanical disruptionmethods that can be used to produce genome fragments include sonicationand shearing.

Random primer whole genome amplification typically produces higheramplification yields and increased representation when intact genomicDNA is used as template compared to fragmented templates. Inapplications of the invention wherein amplification of fragmentedgenomic DNA is desired, it is possible to ligate the fragments togetherto produce concatenated DNA. The concatenated DNA can then be used in awhole genome amplification method such as those set forth previouslyherein. Exemplary conditions that can be used in a genome fragmentconcatenation reaction are described, for example, in WO 03/033724 A1.

In embodiments, in which fragmentation of a target nucleic acid sampleis not desired, the fragments can be modified for use in a method of theinvention. For example, a genomic DNA can be modified to facilitateamplification. An exemplary modification that can facilitateamplification is concatenation of genome fragments to form extendedtemplates that can be efficiently amplified, for example, by randomprimer amplification. Concatenation can be carried out for example bytreating a population of genome fragments with T4 RNA ligase underconditions known in the art such as those described in McCoy et al.,Biochem. 19:635-642 (1980). Concatenation can also be carried out usinga mixture of AP endonuclease, polymerase and ligase. Damaged DNA can berepaired using appropriate enzymes such as the Restorase™ polymerasemixture available from Sigma-Aldrich (R1028). Another modification thatcan be used is the addition of universal tails to genome fragments.Exemplary methods of incorporating universal tails include, withoutlimitation, treatment of fragments with terminal deoxynucleotidestransferase to tail 3′ ends with a mononucleotide such as dGTP.Accordingly, a poly El tail can be added as a universal tail to genomefragments. Poly C, T, U, A, or other nucleotide tails can be added aswell. Universal tails can also be added by treating genome fragmentswith T4 RNA ligase and oligonucleotides having a random 4-mer duplexadapter and universal tail sequence under conditions in which theuniversal tail sequence is added to one or both ends of the genomefragments.

Example X describes methods for amplifying fragments produced bybisulfate treatment of methylated DNA. Those skilled in the art willrecognize that the amplification methods described in Example X can beused for nucleic acid fragment samples of any of a variety ofcompositions and produced by any of a variety of mechanisms. Furtherexamples of DNA fragments useful in the invention include, withoutlimitation, cDNA or degraded genomic DNA, for example, from archivedtissues or cells such as those that are stored formalin-fixed,formaldehyde-fixed, paraffin embedded, polymer embedded, ethanolembedded or by some combination thereof. Fragmented DNA can also beobtained from forensic samples, archeological samples, paleontologicalsamples, mummified samples, petrified samples and other samples thathave experienced decay due to an extended period of time between thedeath of the cell or tissue and analysis of its genomic DNA. A method ofdetecting typable loci of a genome can further include a step ofcontacting genome fragments with a plurality of nucleic acid probeshaving sequences corresponding to the typable loci under conditions inwhich probe-fragment hybrids are formed. A probe used in a method of theinvention can have any of a variety of compositions or sizes, so long asit has the ability to bind to a target nucleic acid with sequencespecificity. Typically, a probe used in the methods is a nucleic acidincluding, for example, one having a native structure or an analogthereof. Exemplary nucleic acid probes that can be used in a method ofthe invention include, without limitation, those set forth above inregard to primers and other nucleic acids useful in the invention. Itwill be further understood that other sequence specific probes can alsobe used in a method of the invention including, for example, peptides,proteins or other polymeric compounds.

Probes of the present invention can be complementary to typable loci orother detection positions that are indicative of the presence of thetypable loci in a representative population of genome fragments. Thus, astep of detecting a typable locus of a genome fragments can include, forexample, detecting the locus itself or detecting another sequence thatis genetically linked or associated. This complementarity need not beperfect. For example, there can be any number of base pair mismatcheswithin a hybridized nucleic acid complex, so long as the mismatches donot prevent formation of a sufficiently stable hybridization complex fordetection under the conditions being used.

Furthermore, nucleic acid probes used in a method of the invention caninclude sequence regions that are not complementary to target sequencesor other sequences present in a particular population of genomefragments. These non-target complementing sequence regions can include,for example, tinker sequences for attaching the probes to a substrate,annealing sites for other nucleic acids such as a primer or otherdesired sequences. A target-complementing sequence region of a nucleicacid probe can have a length that is, for example, at least 10nucleotides in length. Longer target-complementing regions can also beuseful including, without limitation, those that are at least about 15,20, 25, 35, 50, 70, 100, 500, 1000, or 5000 nucleotides in length orlonger. As set forth above, particular embodiments of the inventionprovide the ability to amplify a native genome to produce arepresentative population of relatively small genome fragments. Anon-limiting advantage of detecting typable loci of a genome on smallgenome fragments is that loci that are relatively close can be separatedfor individual detection. Accordingly, in particular embodiments, suchas detection of small target sequences, a target-complementary region ofa nucleic acid probe can be at most about 100, 90, 80, 70, 60, 50, 40,35, 30, 25, 20, or 10 nucleotides in length.

Exemplary target-complementing sequences that are useful in theinvention are set forth below in the context of various detectiontechniques. Those skilled in the art will understand that the probesneed not be limited to use in the particular detection techniqueexemplified but rather can be used in any of a variety of differentdetection techniques as desired for a particular application of theinvention.

A probe used in a method of the invention can further have amodification, for example, to support a particular detection method. Forexample, in embodiments wherein amplification or modification of aparticular probe is not desired, the probe can have a structure that isresistant to modification. As specific examples, a probe can lack a 3′OH group or have a 3′ cap moiety, thereby being inert to modificationwith a polymerase. In particular embodiments, a probe can include adetectable label including, without limitation, one or more of theprimary or secondary nucleic acid labels set forth above. Alternatively,detection can be based on an intrinsic characteristic of the probe,fragment or hybrid such that labeling is not required. Examples ofintrinsic characteristics that can be detected include, but are notlimited to, mass, electrical conductivity, energy absorbance,fluorescence or the like.

Any of a variety of conditions can be used to hybridize probes withgenome fragments including, without limitation, those set forth above inregard to primer annealing to target. In particular embodiments, thehybridization conditions can support modification or replication of theprobe, genome fragment or both. However, depending upon the detectionmethod in which the probe is applied, hybridization conditions need notsupport modification of a probe-fragment hybrid. Accordingly, thepresence of a particular fragment can be determined based on adetectable property of the genome fragment, probe or both. Furtherexemplary hybridization conditions are set forth below in regard toparticular detection methods.

A plurality of genome fragments that is contacted with probes in amethod of the invention can represent all or part of a genome sequence.Accordingly, the complexity of the plurality of genome fragments can beequivalent to the size of the genome from which it was amplified orotherwise produced. For example, a plurality of human genome fragmentsthat are contacted with probes can have a complexity of about 3.1Gigabases which is roughly equivalent to the full length genome. Lowercomplexity representations can also be used. Again using the humangenome as a non-limiting example, a plurality of genome fragments thatare contacted with probes can have a complexity of at least about 2Gigabases, which is a representation of about 60% of the human genome ora complexity of at least about 1 Gigabases, which is a representation ofat least about 30% of the human genome. The complexity of a plurality ofprobes contacted with probes in a method of the invention can be, forexample, at, least, about 0.1 Gigabases, 0.2 Gigabases, 0.5 Gigabases,0.8 Gigabases, 1 Gigabases, 1.5 Gigabases, 2 Gigabases, 2.5 Gigabases, 3Gigabases, 3.5 Gigabases, 4 Gigabases, 4.5 Gigabases, 5 Gigabases ormore.

As higher complexity pluralities of genome fragments are used in amethod of the invention it is typically desired to use larger amounts ofDNA. Accordingly, the amount of DNA in a plurality of genome fragmentsthat is contacted with probes in a method disclosed herein can be atleast about 1 ug, 10 ug, 50 ug, 100 ug, 150 ug, 200 ug, 300 ug, 400 ug,500 ug, 1000 ug or more (ug herein refers to a microgram). A pluralityof genome fragments can be present in a fluid sample at anyconcentration that gives desired results such as a desired level ofsequence-specific hybridization between probes and fragments or amountof loci detected. For example, the concentration of a plurality ofgenome fragments contacted with probes in a method of the invention canbe at least about 0.1 ug/ul, 0.2 ug/ul, 0.5 ug/ul, 0.8 ug/ul, 1 ug/ul,1.5 ug/ul, 2 ug/ul, 5 ug/ul, 10 ug/ul (ul herein refers to a microliter)

The number of probes contacted with a plurality of genome fragments canbe selected based on a desired application of the methods. Exemplaryprobe populations and arrays that can be used include those known in theart and/or set forth herein. The number of different probes that formsequence-specific hybrids with genome fragments can be, for example, atleast about 100, 500, 1000, 5000, 1×10⁴, 5×10⁴, 1×10⁵, 5×10⁵, 1×10⁶,5×10⁶,or more including a number of probes in a population or arrayknown in the art and/or set forth herein.

Following hybridization, non-hybridized nucleic acids can be separatedfrom hybrids, if desired. Single strand nucleic acids and hybrid nucleicacids can be separated based on properties that differ for the twospecies including, for example, size, mass, energy absorbance,fluorescence, electrical conductivity, charge, or affinity forparticular substrates. Exemplary methods that can be used to separatesingle strand nucleic acids and hybrid nucleic acids based on propertiesthat differ for the two species include, but are not limited to, sizeexclusion chromatography, filtration through a membrane having aparticular size cutoff, affinity chromatography, gel electrophoresis,capillary electrophoresis, fluorescent activated cell sorting (FACS),and the like.

In a particular embodiment, separation of single strand nucleic acids,such as probes, targets or both, from hybrid nucleic acids can befacilitated by attachment of the probe or target to a substrate. Anexemplary method including separation of nucleic acids using a solidphase substrate is shown in FIG. 9 and described above. Hybrids formedon the substrate bound nucleic acid can be separated from non-hybridizednucleic acids by physical separation of the substrate from the reactionmixture. Exemplary substrates that can be used for such separationinclude, without limitation, particles such as magnetic beads,Sephadex™, controlled pore glass, agarose or the like; or surfaces suchas glass surfaces, plastic, ceramics and the like. Nucleic acids can beattached to substrates via known linkers and ligands such as those setforth above in regard to nucleic acid secondary labels and using methodsknown in the art. Substrates can be physically separated from a solutionby any of a variety of methods including, for example, magneticattraction, gravity sedimentation, centrifugal sedimentation,filtration, FACS, electrical attraction or the like. Separation can alsobe carried out by manual movement of the substrate, for example, usingthe hands or a robotic device.

A method of the invention can further include a step of detectingtypable loci of probe-genome fragment hybrids. Depending upon theparticular application of the invention, probe-genome fragment hybridscan be detected using a direct detection technique, or alternatively anamplification-based technique, Direct detection techniques include thosein which the level of nucleic acids in probe-fragment hybrids providesthe detected signal, For example, in the case of a hybrid formed at aparticular array location, the signal from the location arising from thecaptured hybrid or its component nucleic acids can be detected withoutamplifying the hybrid or its component nucleic acids. Alternatively,detection can include amplification of the probe or genome fragment orboth to increase the level of nucleic acid that is detected. As setforth below in the context of various exemplary detection techniques, aprobe nucleic acid, genome fragment or both can be labeled. Furthermore,nucleic acids in a probe-fragment hybrid can be labeled prior to, duringor after hybrid formation and detection of typable loci based ondetection of such labels

Accordingly a method of detecting typable loci of a genome can includethe steps of (a) providing an amplified representative population ofgenome fragments that has such typable loci, (b) contacting the genomefragments with a plurality of nucleic acid probes having sequencescorresponding to the typable loci under conditions whereinprobe-fragment hybrids are formed; and (c) directly detecting typableloci of the probe-fragment hybrids.

Generally, detection, whether direct or based on an amplificationtechnique, can be achieved by methods that perceive properties that areintrinsic to nucleic acids or their associated labels. Useful propertiesinclude, for example, those that can be used to distinguish nucleicacids having typable loci from those lacking the loci. Such detectedproperties can be used to distinguish different nucleic acids alone orin combination with other methods such as attachment to discretelocations of a detection array. Exemplary properties upon whichdetection can be based include, but are not limited to, mass, electricalconductivity, energy absorbance, fluorescence or the like.

Detection of fluorescence can be carried out by irradiating a nucleicacid or its label with an excitatory wavelength of radiation anddetecting radiation emitted from a fluorophore therein by methods knownin the art and described for example in Lakowicz, Principles ofFluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999). Afluorophore can be detected based on any of a variety of fluorescencephenomena including, for example, emission wavelength, excitationwavelength, fluorescence resonance energy transfer (FRET) intensity,quenching, anisotropy or lifetime. FRET can be used to identifyhybridization between a first polynucleotide attached to a donorfluorophore and a second polynucleotide attached to an acceptorfluorophore due to transfer of energy from the excited donor to theacceptor. Thus, hybridization can be detected as a shift in wavelengthcaused by reduction of donor emission and appearance of acceptoremission for the hybrid. In addition, fluorescence recovery afterphotobleaching (FRAP) can be used to identify hybridization according tothe increase in fluorescence occurring at a previously photobleachedarray location due to binding of a fluorescently labeled targetpolynucleotide.

Other detection techniques that can be used to perceive or identifynucleic acids having typable loci include, for example, massspectrometry which can be used to perceive a nucleic acid based on itsmass; surface plasmon resonance which can be used to perceive a nucleicacid based on binding to a surface immobilized complementary sequence;absorbance spectroscopy which can be used to perceive a nucleic acidbased on the wavelength of the energy it absorbs; calorimetry which canbe used to perceive a nucleic acid based on changes in temperature ofits environment due to binding to a complementary sequence; electricalconductance or impedance which can be used to perceive a nucleic acidbased on changes in its electrical properties or in the electricalproperties of its environment, magnetic resonance which can be used toperceive a nucleic acid based on presence of magnetic nuclei, or otherknown analytic spectroscopic or chromatographic techniques.

In particular embodiments, typable loci of probe-fragment hybrids can bedetected based on the presence of the probe, fragment or both in thehybrid, without subsequent modification of the hybrid species. Forexample, a pre-labeled fragment having a particular typable locus can beidentified based on presence of the label at a particular array locationwhere a nucleic acid complement of the locus resides.

The invention further provides a method of detecting typable loci of agenome including the steps of (a) providing an amplified representativepopulation of genome fragments having the typable loci; (b) contactingthe genome fragments with a plurality of immobilized nucleic acid probeshaving sequences corresponding to the typable loci under conditionswherein immobilized probe-fragment hybrids are formed; (c) modifying theimmobilized probe-fragment hybrids; and (d) detecting a probe orfragment that has been modified, thereby detecting the typable loci ofthe genome,

In a particular embodiment, arrayed nucleic acid probes can be modifiedwhile hybridized to genome fragments for detection. Such embodiments,include, for example, those utilizing ASPS, SBE, oligonucleotideligation amplification (OLA), extension ligation (GoldenGate™), invadertechnology, probe cleavage or pyrosequencing as described in U.S. Pat.No. 6,355,431 B1, U.S. Ser. No. 10/177,727 and/or below. Thus, theinvention can be carried out in a mode wherein an immobilized probe ismodified instead of a genome fragment captured by a probe.Alternatively, detection can include modification of the genomefragments while hybridized to probes. Exemplary modifications includethose that are catalyzed by an enzyme such as a polymerase. A usefulmodification can be incorporation of one or more nucleotides ornucleotide analogs to a primer hybridized to a template strand, whereinthe primer can be either the probe or genome fragment in aprobe-genome-fragment hybrid. Such a modification can includereplication of all or part of a primed template. Modification leading toreplication of only a part of a template probe or genome fragment willbe understood to be detection without amplification of the templatesince the template is not replicated along its full length.

Extension assays are useful for detection of typable loci. Extensionassays are generally carried out by modifying the 3′ end of a firstnucleic acid when hybridized to a second nucleic acid. The secondnucleic acid can act as a template directing the type of modification,for example, by base pairing interactions that occur duringpolymerase-based extension of the first nucleic acid to incorporate oneor more nucleotide. Polymerase extension assays are particularly useful,for example, due to the relative high-fidelity of polymerases and theirrelative ease of implementation. Extension assays can be carried out tomodify nucleic acid probes that have free 3′ ends, for example, whenbound to a substrate such as an array. Exemplary approaches that can beused include, for example, allele-specific primer extension (ASPS),single base extension (SBE), or pyrosequencing.

In particular embodiments, single base extension (SBE) can be used fordetection of typable loci. An exemplary diagrammatic representation ofSBE is shown in FIG. 2 . Briefly, SBE utilizes an extension probe thathybridizes to a target genome fragment at a location that is proximal oradjacent to a detection position, the detection position beingindicative of a particular typable locus. A polymerase can be used toextend the 3′ end of the probe with a nucleotide analog labeled with adetection label such as those described previously herein. Based on thefidelity of the enzyme, a nucleotide is only incorporated into theextension probe if it is complementary to the detection position in thetarget genome fragment. If desired, the nucleotide can be derivatizedsuch that no further extensions can occur, and thus only a singlenucleotide is added. The presence of the labeled nucleotide in theextended probe can be detected, for example, at a particular location inan array and the added nucleotide identified to determine the identityof the typable locus. SBE can be carried out under known conditions suchas those described in U.S. patent application Ser. No. 09/425,633. Alabeled nucleotide can be detected using methods such as those set forthabove or described elsewhere such as Syvanen et al., Genomics 8:684-692(1990); Syvanen et al., Human Mutation 3:172-179 (1994); U.S. Pat. Nos.5,846,710 and 5,888,819; Pastinen et al., Genomics Res. 7(6):606-614(1997).

A nucleotide analog useful for SBE detection can include adideoxynucleoside-triphosphate (also called deoxynucleotides or ddNTPs,i.e. ddATP, ddTTP, ddCTP and ddGTP), or other nucleotide analogs thatare derivatized to be chain terminating. The use of labeled chainterminating nucleotides is useful, for example, in reactions having morethan one type of dNTP present so as to prevent false positives due toextension beyond the detection position. Exemplary analogs aredideoxy-triphosphate nucleotides (ddNTPs) or acyclo terminators (PerkinElmer, Foster City, Calif.). Generally, a set of nucleotides comprisingddATP, ddCTP, ddGTP and ddTTP can be used, at least one of whichincludes a label. If desired for a particular application, a. set ofnucleotides in which all four are labeled can be used. The labels canall be the same or, alternatively, different nucleotide types can havedifferent labels. As will be appreciated by those in the art, any numberof nucleotides or analogs thereof can be added to a primer, as long as apolymerase enzyme incorporates a particular nucleotide of interest at aninterrogation position that is indicative of a typable locus.

A nucleotide used in an SBE detection method can further include, forexample, a detectable label, which can be either a primary or secondarydetectable label. Any of a variety of the nucleic acid labels set forthpreviously herein can be used in an SBE detection method. The use ofsecondary labels can also facilitate the removal of unextended probes inparticular embodiments.

The solution for SBE can also include an extension enzyme, such as a DNApolymerase. Suitable DNA polymerases include, but are not limited to,the Klenow fragment of DNA polymerase I, SEQUENASE™ 1.0 and SEQUENASE™2.0 (U.S. Biochemical), T5 DNA polymerase, Phi29 DNA polymerase,Thermosequenase™ (Taq with the Tabor-Richardson mutation) and othersknown in the art or described herein. If the nucleotide is complementaryto the base of the detection position of the target sequence, which isadjacent to the extension primer, the extension enzyme will add it tothe extension primer. Thus, the extension primer is modified, i.e.extended, to form a modified primer.

In embodiments where the amount of unextended primer in the reactiongreatly exceeds the resultant extended-labeled primer and the excess ofunextended primer competes with the detection of the labeled primer,unextended primers can be removed. For example, unextended primers canbe removed from SBE reactions that are run with small amounts of DNAtarget. Useful methods for removing unextended primers are set forthherein. Furthermore, single stranded probes can be preferentiallyremoved from an array of probes, leaving double-stranded probe-targethybrids using methods set forth in further detail below such asexonuclease treatment. Such methods can provide increased assaysensitivity and selective detection, for example, by removing backgroundarising from non-template directed probe labeling.

As will be appreciated by those in the art, the configuration of an SBEreaction can take on any of several forms. In particular embodiments,the reaction can be done in solution, and then the newly synthesizedstrands, with the base-specific detectable labels, can be detected. Forexample, they can be directly hybridized to capture probes that arecomplementary to the extension primers, and the presence of the labelcan then be detected. Such a configuration is useful, for example, whengenome fragments are arrayed as capture probes. Alternatively, the SBEreaction can occur on a surface. For example, a genome fragment can becaptured using a first capture probe that hybridizes to a first targetdomain of the fragment, and the reaction can proceed such that the probeis modified as shown in FIG. 2A.

The determination of the base at the detection position can proceed inany of several ways. In a particular embodiment, a mixed reaction can berun with two, three or four different nucleotides, each with a differentlabel. In this embodiment, the label on the probe can be distinguishedfrom non-incorporated labels to determine which nucleotide has beenincorporated into the probe. Alternatively, discrete reactions can berun each with a different labeled nucleotide. This can be done either byusing a single substrate bound probe and sequential reactions, or byexposing the same reaction to multiple substrate-bound probes, thelatter case being shown in FIG. 2A. For example, dATP can be added to aprobe-fragment hybrid, and the generation of a signal evaluated; thedATP can be removed and dTTP added, etc. Alternatively, four arrays canbe used; the first is reacted with dATP, the second with dTTP, etc., andthe presence or absence of a signal evaluated in each array.

Alternatively, a ratiometric analysis can be done; for example, twolabels, “A” and “B”, on two substrates (e.g. two arrays) can bedetected. In this embodiment, two sets of primer extension reactions areperformed, each on two arrays, with each reaction containing a completeset of four chain terminating NTPs. The first reaction contains two “A”labeled nucleotides and two “B” labeled nucleotides (for example, A andC can be “A” labeled, and. G and T can be “B” labeled). The secondreaction also contains the two labels, but switched; for example, A andG are “A” labeled and T and C are “B” labeled. This reaction compositionallows a biallelic marker to be ratiometrically scored; that is, theintensity of the two labels in two different “color” channels on asingle substrate is compared, using data from a set of two hybridizedarrays. For instance, if the marker is A/G, then the first reaction onthe first array is used to calculate a ratiometric genotyping score; ifthe marker is AlC, then the second reaction on the second array is usedfor the calculation; if the marker is G/T, then the second array isused, etc. This concept can be applied to all possible biallelic markercombinations. In this way, scoring a genotype using a single fiberratiometric score can allow a more robust genotyping than scoring agenotype using a comparison of absolute or normalized intensitiesbetween two different arrays.

The SBE reaction exemplified in FIG. 2 , demonstrates an embodiment inwhich four separate reactions are carried out on four separate arraysusing a single label. Further embodiments can include use of more thanone type of label in combination with fewer than four probe populationsor arrays. For example, SBE can be carried out in a two color mode usinga single reaction and a single probe population. In this mode, all fourchain terminating nucleotides can be present with two of the nucleotidesbearing a first type of label and the other two bearing a second type oflabel. The first label can be used for A and C, whereas the second labelis used for G and T (or G and U). This exemplary labeling scheme allowsdetection of almost 80% of naturally occurring human SNPs since the mostabundant human SNPs are A/G and C/T polymorphisms. Those skilled in theart will recognize that other labeling schemes can be used if desired,for example, to conform to the abundance of polymorphisms in aparticular organism or to conform to the desired types of polymorphismsto be detected in a particular application. The use of SBE with multiplelabel types can provide the non-limiting advantage of reducing thenumber of arrays and reactions required to obtain genotyping data.

Single base sequencing (SBS) is an extension assay that can be carriedout as set forth above for SBE with the exception that one or morenon-chain terminating nucleotides are included in the extensionreaction. Thus, in accordance with the invention, one or more non-chainterminating nucleotides can be included in an SBE reaction including,for example, those set forth above.

An exemplary embodiment of SBS is to carry out two separate reactions ontwo separate probe populations. The two separate reactions areadvantageously carried out using a single label; however, if desiredmore than one type of label can be used. The first reaction can include2 different labeled nucleotides that are extendable and capable ofhybridizing to 2 of the 4 naturally occurring nucleotides in the genomicDNA. The second reaction can include 2 different nucleotides, thenucleotides being labeled and capable of hybridizing to the other 2naturally occurring nucleotides in the genomic DNA. Each of the tworeactions can be devoid of the nucleotides found in the other reactionor can include chain terminating analogs of the nucleotides found in theother reaction. By way of example, the first reaction (hot AC reaction)can include dATP-biotin and dCTP-biotin. This first reaction can lackGTP, UTP and TTP. Alternatively, the first reaction can includedideoxyGTP and dideoxyUTP (or dideoxyGTP and dideoxyTTP). Continuingwith the example, the second reaction (hot GU reaction) can includedGTP-biotin and dUTP-biotin (or dGTP-biotin and dTTP-biotin). Thissecond reaction can lack CTP or ATP. Alternatively, the second reactioncan include dideoxyCTP and dideoxyATP. This exemplary labeling schemeallows detection of almost 80% of naturally occurring human SNPs sincethe most abundant human SNPs are A/G and C/T polymorphisms.

ASPE is an extension assay that utilizes extension probes that differ innucleotide composition at their 3′ end. An exemplary diagrammaticrepresentation of ASPE is shown in FIG. 2B. Briefly, ASPE can be carriedout by hybridizing a target genome fragment to an extension probe havinga 3′ sequence portion that is complementary to a detection position anda 5′ portion that is complementary to a sequence that is adjacent to thedetection position. Template directed modification of the 3′ portion ofthe probe, for example, by addition of a labeled nucleotide by apolymerase yields a labeled extension product, but only if the templateincludes the target sequence. The presence of such a labeledprimer-extension product can then be detected, for example, based on itslocation in an array to indicate the presence of a particular typablelocus.

In particular embodiments, ASPS can be carried out with multipleextension probes that have similar 5′ ends such that they annealadjacent to the same detection position in a target genome fragment butdifferent 3′ ends, such that only probes having a 3′ end thatcomplements the detection position are modified by a polymerase. Asshown in FIG. 2B, a probe having a 3′ terminal base that iscomplementary to a particular detection position is referred to as aperfect match (PM) probe for the position, whereas probes that have a 3′terminal mismatch base and are not capable of being extended in an ASPEreaction are mismatch (MM) probes for the position. The presence of thelabeled nucleotide in the PM probe can be detected and the 3′ sequenceof the probe determined to identify a particular typable locus, An ASPEreaction can include 1, 2, or 3 different MM probes, for example, atdiscrete array locations, the number being chosen depending upon thediversity occurring at the particular locus being assayed. For example,two probes can be used to determine which of 2 alleles for a particularlocus are present in a sample, whereas three different probes can beused to distinguish the alleles of a 3-allele locus.

In particular embodiments, ASPE reaction can include a nucleotide analogthat is derivatized to be chain terminating. Thus, a PM probe in aprobe-fragment hybrid can be modified to incorporate a single nucleotideanalog without further extension, Exemplary chain terminating nucleotideanalogs include, without limitation, those set forth above in regard tothe SBE reaction. Furthermore, one or more nucleotides used in an ASPEreaction whether or not they are chain terminating can include adetection label such as those described previously herein. For example,an ASPE reaction can include a single biotin labeled dNTP as exemplifiedin Example III. If desired, more than one nucleotide in an ASPE reactioncan be labeled. For example reaction conditions such as those describedin Example II can be modified to include biotinylated dCTP as well asbiotinylated dGTP and biotinylated dTTP, An ASPE reaction can be carriedout in the presence of all four nucleotides A, C, T, and G or in thepresence of a subset of these nucleotides including, for example, asubset that lacks substantial amounts of one or more of A, C, T or G.

Pyrosequencing is an extension assay that can be used to add one or morenucleotides to a detection position(s); it is similar to SBE except thatidentification of typable loci is based on detection of a reactionproduct, pyrophosphate (PPi), produced during the addition of a dNTP toan extended probe, rather than on a label attached to the nucleotide.One molecule of PPi is produced per dNTP added to the extension primer.That is, by running sequential reactions with each of the nucleotides,and monitoring the reaction products, the identity of the added base isdetermined. Pyrosequencing can be used in the invention using conditionssuch as those described in US 2002/0001801.

In particular embodiments, modification of immobilized probe-fragmenthybrids can include cleavage or degradation of hybrids having one ormore mismatched base pair. As with other modifications set forth herein,conditions can be employed that result in selective modification ofhybrids having one or more mismatch compared to perfectly matchedhybrids. For example, in an ASPE-based detection method, mismatchprobe-fragment hybrids can be selectively cleaved or degraded comparedto perfect match probe-fragment hybrids. For example, a hybrid can becontacted with an agent that is capable of recognizing a base pairmismatch and modifying the mismatched hybrid such as by bond cleavage.Exemplary agents include enzymes that recognize and cleave hybridshaving mismatched base pairs such as a DNA glycosylase, Cell, T4endonuclease VII, T7 endonuclease I, mung bean endonuclease or Mut-y orothers such as those described in Bradley et al., Nucl. Acids Res.32:2632-2641 (2004). Cleavage products produced from mismatched hybridscan be removed, for example, by washing.

Accordingly, a method of the invention can include modifying immobilizedprobe-fragment hybrids using ASPE along with cleavage of mismatchprobe-fragment hybrids. An advantage of using both modification steps incombination is that specificity can be increased compared to use of onlyone of the steps. For example, in cases wherein ASPE detection is used afirst level of specificity is obtained due to differentiation of matchand mismatch primers by the extending polymerase. In cases whereunwanted mismatch primer extension occurs, cleavage of mismatchedhybrids can act to prevent artifact signal due to mismatch probes,thereby increasing assay specificity and sensitivity. Similarly,specificity and sensitivity can be increased by removing artifact signalarising due to mismatch hybrids formed in other detection methods setforth herein such as ligation based assays. Mismatch hybrids can beremoved from solution phase or solid phase immobilized hybrids inaccordance with the methods disclosed herein.

In a particular embodiment, an A SPE reaction can be carried out underconditions in which extension of perfect match probe-fragment hybrids isdriven to completion and substantial amounts of mismatch probe-fragmenthybrids are also extended. For example, in the case of a locus having anA and B allele, the perfect match probe can be designed against thehomozygous allele A forming a perfect hybrid with an AA individual andthe mismatch probe can be designed against the homozygous allele B,forming a perfect hybrid with a BB individual. Accordingly, the role ofthe perfect match and mismatch probe can be reversed depending on thesample under observation. The product of a mismatch extension will haveone mismatch base pair in the extended product and the perfect matchwill not contain a mismatch. Specific removal of the signal generated bythe mismatch probe, while leaving the signal from the perfect matchextension intact can add a second discrimination step to create a largerdistinction between the perfect match and mismatch, creating a morespecific genotyping assay compared to detection based solely onpolymerase-based modification of perfect match probes.

If desired, an immobilized probe that is not part of a probe-fragmenthybrid can be selectively modified compared to a probe-fragment hybrid.Selective modification of non-hybridized probes can be used to increaseassay specificity and sensitivity, for example, by removing probes thatare labeled in a template independent manner during the course of apolymerase extension assay. A particularly useful selective modificationis degradation or cleavage of single stranded probes that are present ina population or array of probes following contact with target fragmentsunder hybridization conditions. Exemplary enzymes that degrade singlestranded nucleic acids include, without limitation, Exonuclease 1 orlambda Exonuclease.

In embodiments utilizing probes with reactive hydroxyls at their 3′ endsand polymerase extension, a useful exonuclease is one thatpreferentially digests single stranded DNA in the 3′ to 5′ detection.Thus, double stranded probe-target hybrids that form under particularassay conditions are preferentially protected from degradation as is the3′ overhang of the target that serves as a template for polymeraseextension of the probe. However, single stranded probes not hybridizedto target under the assay conditions are preferentially degraded.Furthermore, such exonuclease treatment can preferentially degradesingle stranded regions of genome fragments or other nucleic acids incases where the fragments or nucleic acids are retained by an array dueto interaction with non-probe interacting portions of target nucleicacids. Thus, exonuclease treatment can prevent artifacts that may arisedue to a bridged network of 2 or more nucleic acids bound to a probe.Digestion with exonuclease is typically carried out after a probeextension step.

In some embodiments, detection of typable loci can include amplificationof genome-fragment targets following formation of probe-fragmenthybrids, resulting in a significant increase in the number of targetmolecules. Target amplification-based detection techniques can include,for example, the polymerase chain reaction (PCR), strand displacementamplification (SDA), or nucleic acid sequence based amplification(NASBA). Alternatively, rather than amplify the target, alternatetechniques can use the target as a template to replicate a hybridizedprobe, allowing a small number of target molecules to result in a largenumber of signaling probes, that then can be detected. Probeamplification-based strategies include, for example, the ligase chainreaction (LCR), cycling probe technology (CPT), invasive cleavagetechniques such as Invader™ technology, Q-Beta replicase (QβR)technology or sandwich assays. Such techniques can be carried out, forexample, under conditions described in U.S. Ser. No. 60/161,148,09/553,993 and 090/556,463; and U.S. Pat. No. 6,355,431 131, or as setforth below. These techniques are exemplified below, in the context ofgenome fragments used as target nucleic acids that are hybridized toarrayed nucleic acid probes. It will be understood that in suchembodiments genome fragments can be arrayed as probes and hybridized tosynthetic nucleic acid targets.

Detection with oligonucleotide ligation amplification (OLA) involves thetemplate-dependent ligation of two smaller probes into a single longprobe, using a genome-fragment target sequence as the template. In aparticular embodiment, a single-stranded target sequence includes afirst target domain and a second target domain, which are adjacent andcontiguous. A first OLA probe and a second OLA probe can be hybridizedto complementary sequences of the respective target domains. The two OLAprobes are then covalently attached to each other to form a modifiedprobe. In embodiments where the probes hybridize directly adjacent toeach other, covalent linkage can occur via a ligase. In one embodimentone of the ligation probes may be attached to a surface such as an arrayor a particle. In another embodiment both ligation probes may beattached to a surface such as an array or a particle.

Alternatively, an extension ligation (GoldenGate™) assay can be usedwherein hybridized probes are non-contiguous and one or more nucleotidesare added along with one or more agents that join the probes via theadded nucleotides. Exemplary agents include, for example, polymerasesand ligases. If desired, hybrids between modified probes and targets canbe denatured, and the process repeated for amplification leading togeneration of a pool of ligated probes. As above, theseextension-ligation probes can be but need not be attached to a surfacesuch as an array or a particle, Further conditions for extensionligation assay that are useful in the invention are described, forexample, in U.S. Pat. No. 6,355,431 B1 and U.S. application Ser. No.10/177,727,

OLA is referred to as the ligation chain reaction (LCR) whendouble-stranded genome fragment targets are used. In LCR, the targetsequence can be denatured, and two sets of probes added: one set asoutlined above for one strand of the target, and a separate set (i.e.third and fourth primer probe nucleic acids) for the other strand of thetarget. Conditions can be used in which the first and second probeshybridize to the target and are modified to form an extended probe.Following denaturation of the target-modified probe hybrid, the modifiedprobe can be used as a template, in addition to the second targetsequence, for the attachment of the third and fourth probes. Similarly,the ligated third and fourth probes can serve as a template for theattachment of the first and second probes, in addition to the firsttarget strand. In this way, an exponential, rather than just a linear,amplification can occur when the process of denaturation and ligation isrepeated.

The modified OLA probe product can be detected in any of a variety ofways. In a particular embodiment, a template-directed probe modificationreaction can be carried out in solution and the modified probehybridized to a capture probe in an array. A capture probe is generallycomplementary to at least a portion of the modified OLA probe. In anexemplary embodiment, the first OLA probe can include a detectable labeland the second OLA probe can be substantially complementary to thecapture probe. A non-limiting advantage of this embodiment is thatartifacts due to the presence of labeled probes that are not modified inthe assay are minimized because the unmodified probes do not include thecomplementary sequence that is hybridized by the capture probe. An OLAdetection technique can also include a step of removing unmodifiedlabeled probes from a reaction mixture prior to contacting the reactionmixture with a capture probe as described for example in U.S. Pat. No.6,355,431 B1.

Alternatively, a genome fragment target can be immobilized on asolid-phase surface and a reaction to modify hybridized OLA probesperformed on the solid phase surface. Unmodified probes can be removedby washing under appropriate stringency. The modified probes can then beeluted from the genome fragment target using denaturing conditions, suchas, 0.1 N NaOH, and detected as described herein. Other conditions inwhich a genome fragment can be detected when used as a target sequencein an OLA technique include, for example, those described in U.S. Pat.Nos. 6,355,431 B1, 5,185,243, 5,679,524 and 5,573,907; EP 0 320 308 B1;EP 0 336 731 B1; EP 0 439 182 B1; WO 90/01069; WO 89/12696; WO 97/31256;and WO 89/09835, and U.S. Ser. Nos. 60/078,102 and 60/073,011.

Typable loci can be detected in a method of the invention using rollingcircle amplification (RCA). In a first embodiment, a single probe can behybridized to a genome fragment target such that the probe iscircularized while hybridized to the target. Each terminus of the probehybridizes adjacently on the target nucleic acid and addition of apolymerase results in extension of the circular probe. However, sincethe probe has no terminus, the polymerase continues to extend the proberepeatedly. This results in amplification of the circular probe.Following RCA the amplified circular probe can be detected. This can beaccomplished in a variety of ways; for example, the primer can belabeled or the polymerase can incorporate labeled nucleotides andlabeled product detected by a capture probe in a detection array.Rolling-circle amplification can be carried out under conditions such asthose generally described in Bailer et al. (1998) Nuc. Acids Res.26:5073-5078; Barmy, F. (1991) Proc. Natl. Acad. Sci. USA 88:189-193;and Lizardi et al. (1998) Nat Genet. 19:225-232.

Furthermore, rolling circle probes used in the invention can havestructural features that render them unable to be replicated when notannealed to a target. For example, one or both of the termini thatanneal to the target can have a sequence that forms an intramolecularstem structure, such as a hairpin structure. The stem structure can bemade of a sequence that allows the open circle probe to be circularizedwhen hybridized to a legitimate target sequence but results ininactivation of uncircularized open circle probes. This inactivationreduces or eliminates the ability of the open circle probe to primesynthesis of a modified probe in a detection assay or to serve as atemplate for rolling circle amplification. Exemplary probes capable offorming intramolecular stem structures and methods for their use whichcan be used in the invention are described in U.S. Pat. No. 6,573,051.

In another embodiment, detection can include OLA followed by RCA. Inthis embodiment, an immobilized primer can be contacted with a genomefragment target. Complementary sequences will hybridize with each otherresulting in an immobilized duplex. A second primer can also becontacted with the target nucleic acid. The second primer hybridizes tothe target nucleic acid adjacent to the first primer. An OLA reactioncan be carried out to attach the first and second primer as a modifiedprimer product, for example, as described above. The genome fragment canthen be removed and the immobilized modified primer product, hybridizedwith an RCA probe that is complementary to the modified primer productbut not the unmodified immobilized primer, An RCA reaction can then beperformed.

In a particular embodiment, a padlock probe can be used both for OLA andas the circular template for RCA. Each terminus of the padlock probe cancontain a sequence complementary to a genome fragment target. Morespecifically, the first end of the padlock probe can be substantiallycomplementary to a first target domain, and the second end of the RCAprobe can be substantially complementary to a second target domain,adjacent to the first domain. Hybridization of the padlock probe to thegenome fragment target results in the formation of a hybridizationcomplex, Ligation of the discrete ends of a single oligonucleotideresults in the formation of a modified hybridization complex containinga circular probe that acts as an RCA template complex. Addition of apolymerase to the RCA template complex can allow formation of anamplified product nucleic acid. Following RCA, the amplified productnucleic acid can be detected, for example, by hybridization to an arrayeither directly or indirectly and an associated label detected.

A padlock probe used in the invention can further include othercharacteristics such as an adaptor sequence, restriction site forcleaving concatamers, a label sequence, or a priming site for primingthe RCA reaction as described, for example, in U.S. Pat. No. 6,355,431B1. This same patent also describes padlock probe methods that can beused to detect typable loci of genome fragment targets in a method ofthe invention.

A variation of LCR that can be used to detect typable loci in a methodof the invention utilizes chemical ligation under conditions such asthose described in U.S. Pat. Nos. 5,616,464 and 5,767,259. In thisembodiment, similar to enzymatic modification, a pair of probes can beutilized, wherein the first probe is substantially complementary to afirst domain of a target genome fragment and the second probe issubstantially complementary to an adjacent second domain of the target.Each probe can include a portion that acts as a “side chain” that formsone half of a non-covalent stern structure between the probes ratherthan binding the target sequence. Particular embodiments utilizesubstantially complementary nucleic acids as the side chains. Thus, uponhybridization of the probes to the target sequence, the side chains ofthe probes are brought into spatial proximity. At least one of the sidechains can include an activatable cross-linking agent, generallycovalently attached to the side chain that upon activation, results in achemical cross-link or chemical ligation with the adjacent probe. Theactivatable group can include any moiety that will allow cross-linkingof the side chains, and include groups activated chemically,photonically or thermally, such as photoactivatable groups. In someembodiments a single activatable group on one of the side chains isenough to result in cross-linking via interaction to a functional groupon the other side chain; in alternate embodiments, activatable groupscan be included on each side chain. One or both of the probes can belabeled

Once a hybridization complex is formed, and the cross-linking agent hasbeen activated such that the probes have been covalently attached toeach other, the reaction can be subjected to conditions to allow for thedisassociation of the hybridization complex, thus freeing up the targetto serve as a template for the next ligation or cross-linking. In thisway, signal amplification can occur, and the cross-linked products canbe detected, for example, by hybridization to an array either directlyor indirectly and an associated label detected.

In particular embodiments, amplification-based detection can be achievedusing invasive cleavage technology. Using such an approach, a genomefragment target can be hybridized to two distinct probes. The two probesare an invader probe, which is substantially complementary to a firstportion of the genome fragment target, and a signal probe, which has a3′ end substantially complementary to a sequence having a detectionposition and a 5′ non-complementary end which can form a single-strandedtail. The tail can include a detection sequence and typically alsocontains at least one detectable label. However, since a detectionsequence in a signal probe can function as a target sequence for acapture probe, sandwich configurations utilizing label probes can beused as described herein and the signal probe need not include adetectable label.

Hybridization of the invader and signal probes near or adjacent to oneanother on a gnome fragment target can form any of several structuresuseful for detection of the probe-fragment hybrid. For example, a forkedcleavage structure can form, thereby providing a substrate for anuclease which cleaves the detection sequence from the signal probe. Thesite of cleavage is controlled by the distance or overlap between the 3′end of the invader probe and the downstream fork of the signal probe.Therefore, neither oligonucleotide is cleaved when misaligned or whenunattached to a genome fragment target.

In particular embodiments, a thermostable nuclease that recognizes theforked cleavage structure and catalyzes release of the tail can be used,thereby allowing thermal cycling of the cleavage reaction and amplified,if desired. Exemplary nucleases that can be used include, withoutlimitation, those derived from Thermus aquaticus, Thermus flavus, orThermus thermophilus; those described in U.S. Pat. Nos. 5,719,028 and5,843,669, or Flap endonucleases (FENs) as described, for example, inU.S. Pat. No. 5,843,669 and Lyamichev et al., Nature Biotechnology17:292-297 (1999).

If desired, the 3′ portion of a cleaved signal probe can be extracted,for example, by binding to a solid-phase capture tag such as bead boundstreptavidin, or by crosslinking through a capture tag to produceaggregates. The 5′ detection sequence of a signal probe, can be detectedusing methods set forth below such as hybridization to a probe on anarray. Invasive cleavage technology can further be used in the inventionusing conditions and detection methods described, for example, in U.S.Pat. Nos. 6,355,431; 5,846,717; 5,614,402; 5,719,028; 5,541,311; or5,843,669.

A further amplification-based detection technique that can be used todetect typable loci is cycling probe technology (CPT). A CPT probe caninclude two probe sequences separated by a scissile linkage. The CPTprobe is substantially complementary to a genome fragment targetsequence and thus will hybridize to it to form a probe-fragment hybrid.The CPT probe can be hybridized to a genome fragment target in a methodof the invention. Typically the temperature and probe sequence areselected such that the primary probe will bind and shorter cleavedportions of the primary probe will dissociate. Depending upon theparticular application, CPT can be done in solution, or either thetarget or scissile probe can be attached to a solid support. Aprobe-fragment hybrid formed in the methods can be subjected to cleavageconditions which cause the scissile linkage to be selectively cleaved,without cleaving the target sequence, thereby separating the two probesequences. The two probe sequences can then be disassociated from thetarget. In particular embodiments, excess probe can be used and thereaction allowed to be repeated any number of times such that theeffective amount of cleaved probe is amplified.

Any linkage within a CPT probe that can be selectively cleaved when theprobe is part of a hybridization complex, that is, when adouble-stranded complex is formed can be used as a scissile linkage. Anyof a variety of scissile linkages can be used in the inventionincluding, for example, RNA which can be cleaved when in a DNA:RNAhybrid by various double-stranded nucleases such as ribonucleases. Suchnucleases will selectively nick or excise RNA nucleosides from a RNA:DNAhybridization complex rather than DNA in such a hybrid or singlestranded DNA. Further examples of scissile linkages and cleaving agentsthat can be used in the invention are described in U.S. Pat. No.6,355,431 B1 and references cited therein.

Upon completion of a CPT cleavage reaction, the uncleaned scissileprobes can be removed or neutralized prior to detection of cleavedprobes to avoid false positive signals, if desired. This can be done inany of a variety of ways including, for example, attachment of theprobes to a solid support prior to cleavage such that following the CPTreaction, cleaved probes that have been released into solution can bephysically separated from uncleaned probes remaining on the support.Uncleaved and cleaved probes can also be separated based on differencesin length, capture of a particular binding label or sequence using, forexample, methods described in U.S. Pat. No. 6,355,431.

Cleaved probes produced by a CPT reaction can be detected using methodssuch as hybridization to an array or other methods set forth herein. Forexample, a cleaved probe can be bound to a capture probe, eitherdirectly or indirectly, and an associated label detected. CPT technologycan be carried out under conditions described, for example, in U.S. Pat.Nos. 5,011,769; 5,403,711; 5,660,988; and 4,876,187, and PCT publishedapplications WO 95/05480; WO 95/1416, and WO 95/00667, and U.S. Ser. No.09/014,304.

In particular embodiments, CPT with a probe containing a scissilelinkage can be used to detect mismatches, as is generally described inU.S. Pat. No. 5,660,988, and WO 95/14106. In such embodiments, thesequence of the scissile linkage can be placed at a position within alonger sequence that corresponds to a particular sequence to bedetected, i.e. the area of a putative mismatch. In some embodiments ofmismatch detection, the rate of generation of released fragments is suchthat the methods provide, essentially, a yes/no result, whereby thedetection of virtually any released fragment indicates the presence of adesired typable locus. Alternatively or additionally, the final amountof cleaved fragments can be quantified to indicate the presence orabsence of a typable locus.

Typable loci of probe-fragment hybrids can also be detected in a methodof the invention using a sandwich assay. A sandwich assay is anamplification-based technique in which multiple probes, typicallylabeled, are bound to a single genome fragment target. In an exemplaryembodiment a genome fragment target can be bound to a solid substratevia a complementary capture probe. Typically, a unique capture probewill be present for each typable locus sequence to be detected. In thecase of a bead array, each bead can have one of the unique captureprobes. If desired, capture extender probes can be used, that allow auniversal surface to have a single type of capture probe that can beused to detect multiple target sequences. Capture extender probesinclude a first portion that will hybridize to all or part of thecapture probe, and a second portion that will hybridize to a firstportion of the target sequence to be detected. Accordingly customizedsoluble probes can be generated, which as will be appreciated by thosein the art can simplify and reduce costs in many applications of theinvention. In particular embodiments, two capture extender probes can beused. This can provide, a non-limiting advantage of stabilizing assaycomplexes, for example, when a target sequence to be detected is large,or when large amplifier probes (particularly branched or dendrimeramplifier probes) are used.

Once a genome fragment target has been bound to a solid substrate, suchas a bead, via a capture probe, an amplifier probe can be hybridized tothe fragment to form a probe-fragment hybrid. Exemplary amplifier probesthat can be used in a method of the invention and conditions for theiruse in sandwich assays are described in U.S. Pat. No. 6,355,431.Briefly, an amplifier probe is a nucleic acid having at least one probesequence, and at least one amplification sequence. A first probesequence of an amplifier probe can be used, either directly orindirectly, to hybridize to a genome fragment target sequence. Anamplification sequence of an amplifier probe can be any of a variety ofsequences that are used, either directly or indirectly, to bind to afirst portion of a label probe. Typically an amplifier probe willinclude a plurality of amplification sequences. The amplificationsequences can be linked to each other in a variety of ways including,for example, covalently linked directly to each other, or to interveningsequences or chemical moieties.

Label probes comprising detectable labels can hybridize to genomefragments thereby firming probe-fragment hybrids and the labels can bedetected to determine the presence of typable loci. The amplificationsequences of the amplifier probe can be used, either directly orindirectly, to bind to a label probe to allow detection. Detection ofthe amplification reactions of the invention, including the directdetection of amplification products and indirect detection utilizinglabel probes (i.e. sandwich assays), can be done by detecting assaycomplexes having labels. Exemplary methods for using a sandwich assayand associated nucleic acids that can be used in the present inventionare further described in U.S. Ser. No. 60/073,011 and in U.S. Pat. Nos.6,355,431; 5,681,702; 5,597,909; 5,545,730; 5,594,117; 5,591,584;5,571,670; 5,580,731; 5,571,670; 5,591,584; 5,624,802; 5,635,352;5,594,118; 5,359,100; 5,124,246 and 5,681,697.

Depending upon a particular application of the methods of the invention,the detection techniques set forth above can be used to detect primarygenome fragment targets or to detect targets in an amplifiedrepresentative population of genome fragments.

In particular embodiments, it can be desirable to remove unextended orunreacted nucleic acids from a reaction mixture prior to detection sinceunextended or unreacted primers can often compete with the modifiedprobes during detection, thereby diminishing the signal, Theconcentration of the unmodified probes relative to modified probes canoften be relatively high, for example in embodiments where a largeexcess of probe is used. Accordingly, a number of different techniquescan be used to facilitate the removal of unextended primers. Exemplarymethods that can be used to remove unextended primers include, forexample, those described in U.S. Pat. No. 6,355,431.

As set forth above, the invention can be used to detect one or moretypable In particular, the invention is well suited to detection of aplurality of typable loci because the methods allow individual loci tobe distinguished within large and complex pluralities. Individualtypable loci can be distinguished in the invention based on separationof the loci into individual genome fragments, formation ofprobe-fragment hybrids and detection of physically separatedprobe-fragment hybrids. Physical separation of probe-fragment hybridscan be achieved in the invention by binding the hybrids or theircomponents to one or more substrates. In particular embodiments, aprobe-fragment hybrid can be distinguished from other probes andfragments in a plurality based on the physical location of the hybrid onthe surface of a substrate such as an array. A probe-fragment hybrid canalso be bound to a particle. Particles can be discretely detected basedon their location and distinguished from other probes and fragmentsaccording to discrete detection of the particle on a surface such as abead array or in a fluid sample such as a fluid stream in a flowcytometer. Exemplary formats for distinguishing probe-fragment hybridsfor detection of individual typable loci are set forth in further detailbelow,

Detection of typable loci in an amplified representative population ofgenome fragments can employ arrays, In embodiments where relativelylarge numbers of loci are to be detected, arrays are preferably highdensity arrays. Exemplary microarrays that can be used in the inventioninclude, without limitation, those described in Butte, Nature ReviewsDrug Discov. 1:951-60 (2002) or U.S. Pat. Nos. 5,429,807; 5,436,327;5,561,071; 5,583,211; 5,658,734; 5,837,858; 5,874,219; 5,919,523;6,136,269; 6,287,768; 6,287,776; 6,288,220; 6,297,006; 6,291,193;6,346,413; 6,416,949; 6,482,591; 6,514,751 and 6,610,482; and WO93/17126; WO 95/11995; WO 95/35505; EP 742 287; and EP 799 897. Furtherexamples of array formats that are useful in the invention are describedin U.S. Pat. No. 6,355,431 BI, US 2002/0102578 and PCT Publication No.WO 00/63437, Exemplary formats that can be used in the invention todistinguish beads in a fluid sample using microfluidic devices aredescribed, for example, in U.S. Pat. No. 6,524,793. Commerciallyavailable fluid formats for distinguishing beads include, for example,those used in xMAP™ technologies from Luminex or MPSS™ methods from LynxTherapeutics. Various techniques and technologies may be used forsynthesizing arrays of biological materials on or in a substrate orsupport to form microarrays. For example, Affymetrix®, GeneChip® arrayscan be synthesized in accordance with techniques sometimes referred toas VLSIPS™ (Very Large Scale Immobilized Polymer Synthesis)technologies. Some aspects of VLSIPS™ and other microarray and polymer(including protein) array manufacturing methods and techniques have beendescribed in U.S. patent Ser. No. 09/536,841, international PublicationNo. WO 00/58516; U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743,5,324,633, 5,445,934, 5,744,305, 5,384,261, 5,405,783, 5,424,186,5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215, 5,571,639,5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734, 5,795,716,5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,936,324, 5,968,740,5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860, 6,040,193,6,090,555, 6,136,269, 6,269,846, 6,022,963, 6,083,697, 6,291,183,6,309,831 and 6,428,752; and in PCT Applications Nos. PCT/US99/00730(International Publication No. WO 99/36760) and PCT/US01/04285.

Using VLSIPS™, a GeneChip array can be manufactured by reacting thehydroxylated surface of a 5-inch square quartz wafer with silane.Linkers can then be attached to the silane molecules. The distancebetween these silane molecules determines the probes' packing density,allowing arrays to hold over 500,000 probe locations, or features,within a mere 1.28 square centimeters. Millions of identical DNAmolecules can be synthesized at each feature using a photolithographicprocess in which masks, carrying 18 to 20 square micron windows thatcorrespond to the dimensions of individual features, are placed over thecoated wafer. When ultraviolet light is shone over the mask in the firststep of synthesis, the exposed linkers become deprotected and areavailable for nucleotide coupling. Once the desired features have beenactivated, a solution containing a single type of deoxynucleotide with aremovable protection group can be flushed over the wafer's surface. Thenucleotide attaches to the activated linkers, initiating the synthesisprocess. A capping step can be used to truncate unreacted linkers (orpolynucleotides in subsequent step). In the next synthesis step, anothermask can be placed over the wafer to allow the next round ofdeprotection and coupling. The process is repeated until the probesreach their full length, usually 25 nucleotides. However, probes havingother lengths such as those set forth elsewhere herein can also beattached at each feature. Once the synthesis is complete, the wafers canbe deprotected, diced, and the resulting individual arrays can bepackaged in flowcell cartridges.

A spotted array can also be used in a method of the invention. Anexemplary spotted array is a CodeLink™ Array available from AmershamBiosciences. CodeLink™ Activated Slides are coated with a long-chain,hydrophilic polymer containing amine-reactive groups. This polymer iscovalently crosslinked to itself and to the surface of the slide. Probeattachment can be accomplished through covalent interaction between theamine-modified 5′ end of the oligonucleotide probe and the aminereactive groups present in the polymer. Probes can be attached atdiscrete locations using spotting pens. Useful pens are stainless steelcapillary pens that are individually spring-loaded. Pen load volumes canbe less than about 200 nL with a. delivery volume of about 0.1 nL orless. Such pens can be used to create features having a spot diameterof, for example, about 140-160 μm. In a preferred embodiment, nucleicacid probes at each spotted feature can be 30 nucleotides long, However,probes having other lengths such as those set forth elsewhere herein canalso be attached at each spot.

An array that is useful in the invention can also be manufactured usinginkjet printing methods such as SurePrint™ Technology available fromAglient Technologies. Such methods can be used to synthesizeoligonucleotide probes in situ or to attach pre-synthesized probeshaving moieties that are reactive with a substrate surface. A printedmicroarray can contain 22,575 features on a surface having standardslide dimensions (about 1 inch by 3 inches). Typically, the printedprobes are 25 or 60 nucleotides in length. However, probes having otherlengths such as those set forth elsewhere herein can also be printed ateach location.

For several of the embodiments described herein nucleic acid probes areattached to substrates such that they have a free 3′ end formodification by enzymes or other agents. Those skilled in the art willrecognize that methods exemplified above in regard to synthesis ofnucleic acids in the 3′ to 5′ direction can be modified to producenucleic acids having free 3′ ends. For example, synthetic methods knownin the art for synthesizing nucleic acids in the 5′ to 3′ direction andhaving 5′ attachments to solid supports can be used in an inkjetprinting or photolithographic method. Furthermore, in situ inversion ofsubstrate attached nucleic acids can be carried out such that 3′substrate-attached nucleic acids become attach to the substrate at their5′ end and detached at their 3′ end, In situ inversion can be carriedout according to methods known in the art such as those described inKwiatkowski et al., Nucl. Acids Res. 27:4710-4714 (1999).

An exemplary high density array is an array of arrays or a compositearray having a plurality of individual arrays that is configured toallow processing of multiple samples. Such arrays allow multiplexdetection of typable loci. Exemplary composite arrays that can be usedin the invention, for example, in multiplex detection formats aredescribed in U.S. Pat. No. 6,429,027 and. US 2002/0102578. In particularembodiments, each individual array can be present within each well of amicrotiter plate. Thus, depending on the size of the microtiter plateand the size of the individual array, very high numbers of assays can berun simultaneously; for example, using individual arrays of 2,000 and a96 well microtiter plate, 192,000 assays can be performed in parallel;the same number of arrays in each well of a 384 microtiter plate yields768,000 simultaneous assays, and in a 1536 microtiter plate gives3,072,000 assays.

In particular embodiments, nucleic acids useful in detecting typableloci of a genome can be attached to particles that are arrayed orotherwise spatially distinguished. Exemplary particles includemicrospheres or beads. However, particles used in the invention need notbe spherical. Rather particles having other shapes including, but notlimited to, disks, plates, chips, slivers or irregular shapes can beused. In addition, particles used in the invention can be porous, thusincreasing the surface area available for attachment or assay ofprobe-fragment hybrids. Particle sizes can range, for example, fromnanometers such as about 100 nm beads, to millimeters, such as about 1mm beads, with particles of intermediate size such as at most about 0.2micron, 0.5 micron, 5 micron or 200 microns being useful. Thecomposition of the beads can vary depending, for example, on theapplication of the invention or the method of synthesis. Suitable beadcompositions include, but are not limited to, those used in peptide,nucleic acid and organic moiety synthesis, such as plastics, ceramics,glass, polystyrene, methylstyrene, acrylic polymers, paramagneticmaterials, thoria sot, carbon graphite, titanium dioxide, latex orcross-linked dextrans such as Sepharose™, cellulose, nylon, cross-linkedmicelles or Teflon™. Useful particles are described, for example, inMicrosphere Detection Guide from Bangs Laboratories, Fishers Ind.

Several embodiments of array-based detection in the invention areexemplified below for beads or microspheres. Those skilled in the artwill recognize that particles of other shapes and sizes, such as thoseset forth above, can be used in place of beads or microspheresexemplified for these embodiments.

Each particle used for detection of typable loci in a population ofgenome fragments can include an associated capture probe. However, ifdesired, one or more particles can be included in an array or populationof particles that do not contain a capture probe. A capture probe can beany molecule or material that directly or indirectly binds a nucleicacid having a target sequence such as a typable locus. A capture probecan be, for example, a nucleic acid that has a sequence that hybridizesto a complementary nucleic acid or another molecule that binds to anucleic acid in a sequence-specific fashion.

In a particular embodiment, each bead or other array location can have asingle type of capture probe. However, a plurality of probes can beattached to each bead if desired. For example, a bead or other arraylocation can have two or more probes that anneal to different portionsof the same genome fragment. The probes can anneal to adjacent locationsor at locations that are separated front each other on the capturedtarget nucleic acid. Use of this multiple probe capture embodiment canincrease specificity of detection compared to the use of only one of theprobes. Thus, in cases where smaller probes are desired a multiple probestrategy can be employed to provide specificity comparable toembodiments where longer probes are utilized. Similarly, a subpopulationof more than one microsphere containing a particular capture probe canbe used to detect typable loci of a genome in the invention. Thus,redundancy can be built into the assay system by the use ofsubpopulations of microspheres for particular probes.

In some embodiments, polymer probes such as nucleic acids or peptidescan be synthesized by sequential addition of monomer units directly on asolid support used in an array such as a bead or slide surface. Methodsknown in the art for synthesis of a variety of different chemicalcompounds on solid supports can be used in the invention, such asmethods for solid phase synthesis of peptides, organic moieties, andnucleic acids. Alternatively probes can be synthesized first, and thencovalently attached to a solid support. Probes can be attached tofunctional groups on a solid support. Functionalized solid supports canbe produced by methods known in the art and, if desired, obtained fromany of several commercial suppliers for beads and other supports havingsurface chemistries that facilitate the attachment of a desiredfunctionality by a user. Exemplary surface chemistries that are usefulin the invention include, but are not limited to, amino groups such asaliphatic and aromatic amines, carboxylic acids, aldehydes, amides,chloromethyl groups, hydrazide, hydroxyl groups, sulfonates or sulfates.If desired, a probe can be attached to a solid support via a chemicallinker.

Such a linker can have characteristics that provide, for example, stableattachment, reversible attachment, sufficient flexibility to allowdesired interaction with a genome fragment having a typable locus to bedetected, or to avoid undesirable binding reactions. Further exemplarymethods that can be used in the invention to attach polymer probes to asolid support are described in Pease et al., Proc. Natl. Acad. Sci. USA91(11):5022-5026 (1994); Khrapko et al., Mol Biol (Mosk) (USSR)25:718-730 (1991); Stimpson et al., Proc. Natl. Acad, Sci, USA92:6379-6383 (1995) or Guo et al., Nucleic Acids Res. 22:5456-5465(1994).

Generally, an array of arrays can be configured in any of several ways.In a particular embodiment, as is more fully described below, a onecomponent system can be used. That is, a first substrate having aplurality of assay locations, such as a microliter plate, can beconfigured such that each assay location contains an individual array.Thus, the assay location and the array location can be the same. Forexample, the plastic material of a microliter plate can be formed tocontain a plurality of bead wells in the bottom of each of the assaywells. Beads containing the capture probes of the invention can then beloaded into the bead wells in each assay location as is more fullydescribed below.

Alternatively, a two component system can be used. In this embodiment,individual arrays can be formed on a second substrate, which then can befitted or dipped into the first microliter plate substrate. A particularembodiment utilizes fiber optic bundles as individual arrays, generallywith bead wells etched into one surface of each individual fiber, suchthat the beads containing the capture probes are loaded onto the end ofthe fiber optic bundle. The composite array thus includes a number ofindividual arrays that are configured to fit within the wells of amicrotiter plate.

Accordingly, the present invention provides a composite array having atleast a first substrate with a surface having a plurality of assaylocations. Any of a variety of arrays having a plurality of candidateagents in an array format can be used in the invention. The size of anarray used in the invention can vary depending on the probe compositionand desired use of the array. Arrays containing from about 2 differentprobes to many millions can be made, with very large fiber optic arraysbeing possible. Generally, an array can have from two to as many as abillion or more array locations per square cm. An array location can be,for example, an area on a surface to which a probe or population ofsimilar probes are attached or a particle. In the case of a particle,its array location can be a fixed coordinate on a substrate to which itis attached or associated, or a relative coordinate compared tolocations of one or more other reference particles in a fluid samplesuch as a stream passing through a flow cytometer. Very high densityarrays are useful in the invention including, for example, those havingfrom about 10,000,000 array locations/cm² to about 2,000,000,000 arraylocations/cm² or from about 100,000,000 array locations/cm² to about1,000,000,000 array locations/cm². High density arrays can also be usedincluding, for example, those in the range from about 100,000 arraylocations/cm² to about 10,000,000 array locations/cm² or about 1,000,000array locations/cm² to about 5,000,000 array locations/cm². Moderatedensity arrays useful in the invention can range from about 10,000 arraylocations/cm² to about 100,000 array locations/cm², or from about 20,000array locations/cm² to about 50,000 array locations/cm². Low densityarrays are generally less than 10,000 particles/cm² with from about1,000 array locations/cm² to about 5,000 array locations/cm² beinguseful in particular embodiments. Very low density arrays having lessthan 1,000 array locations/cm², from about 10 array locations/cm² toabout 1000 array locations/cm², or from about 100 array locations/cm² toabout 500 array locations/cm² are also useful in some applications. Themethods of the invention need not be performed in array format, forexample, in embodiments in which one or a small number of loci are to bedetected. If desired, arrays having multiple substrates can be used,including, for example substrates having different or identicalcompositions. Thus for example, large arrays can include a plurality ofsmaller substrates,

For some applications the number of individual arrays is set by the sizeof the microliter plate used; thus, 96 well, 384 well and 1536 wellmicroliter plates utilize composite arrays comprising 96, 384 and 1536individual arrays. As will be appreciated by those in the art, eachmicroliter well need not contain an individual array. It should be notedthat composite arrays can include individual arrays that are identical,similar or different. For example, a composite array having 96 similararrays can be used in applications where it is desired to determine thepresence or absence of the same 2,000 typable loci for 96 differentsamples. Alternatively, a composite array having 96 different arrays,each with 2,000 different probes, can be used in applications where itis desired to determine the presence or absence of 192,000 typable locifor a single sample. Alternative combinations, where rows, columns orother portions of a microtiter formatted array are the same can be used,for example, in cases where redundancy is desired. As will beappreciated by those in the art, there are a variety of ways toconfigure the system. In addition, the random nature of the arrays canmean that the same population of beads can be added to two differentsurfaces, resulting in substantially similar but perhaps not identicalarrays.

A substrate used in an array of the invention can be made from anymaterial that can be modified to contain discrete individual sites andis amenable to at least one detection method. In embodiments wherearrays of particles are used a material that is capable of attaching orassociating with one or more type of particles can be used. Usefulsubstrates include, but are not limited to, glass; modified glass;functionalized glass; plastics such as acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, Teflon, or the like; polysaccharides;nylon; nitrocellulose; resins; silica; silica-based materials such assilicon or modified silicon; carbon; metal; inorganic glass; opticalfiber bundles, or any of a variety of other polymers. Useful substratesinclude those that allow optical detection, for example, by beingtranslucent to energy of a desired detection wavelength and/or do notthemselves appreciably fluoresce in a desired detection wavelength.

Generally a substrate used for an array of the invention has a flat orplanar surface. However, other configurations of substrates can be usedas well. For example, three dimensional configurations can be used byembedding an array, such as a bead array in a porous material, such as ablock of plastic, that allows sample access to the array locations anduse of a confocal microscope for detection. Similarly, assay locationscan be placed on the inside surface of a tube, for flow-through sampleanalysis. Exemplary substrates that are useful in the invention include,but are not limited to, optical fiber bundles, or flat planar substratessuch as glass, polystyrene or other plastics and acrylics.

The surface of a substrate can include a plurality of individual arraylocations that are physically separated from each other. For example,physical separation can be due to the presence of assay wells, such asin a microtiter plate. Other barriers that can be used to physicallyseparate array locations include, for example, hydrophobic regions thatwill deter flow of aqueous solvents or hydrophilic regions that willdeter flow of apolar or hydrophobic solvents.

Array locations that are physically separated from each other form assaylocations. An assay location can include an array of probes and providea vessel for holding a fluid such that the fluid contacts the probes.For example, a fluid containing genome fragments can be contacted withprobes under hybridization conditions set forth herein or known in theart. Similarly, a wash fluid or fluid containing other reagents oranalytes described herein can be contacted with an array of probes whenplaced in an assay location. An assay location can be enclosed, ifdesired. Exemplary enclosures include, without limitation, a cassette,enclosed well, or a slide surface enclosed by a gasket or membrane orboth. Further exemplary enclosures that are useful in the invention aredescribed in WO 02/00336, US Pat. App. Pub, 02/0102578 or the referencescited previously herein in regard to different types of arrays.

An assay location can also be the interior of a flow cell. An array ofprobes can be placed at an interior surface of the flow cell and a fluidintroduced by flowing into the cell. A flow cell useful in the inventioncan be a capillary gap flow cell. A capillary gap flow cell has asufficiently narrow interior dimension and openings such that a fluidcan be retained in the cell by capillary action and subsequentlydisplaced by positive pressure exerted at an opening by a second fluid.Positive pressure can be provided, for example, by gravity flow. Anexemplary capillary flow cell that is useful in the invention is oneformed between the surface of a slide-based array such as a BeadChiparray (Illumina, Inc., San Diego Calif.) and a Coverplate(ThermoShandon, Inc., Pittsburgh, Pa.). Another useful capillary gapflow cell is that used in the GenePaint™ flow through system availablefrom Tecan (Maennedorf, Switzerland). Accordingly, the inventionprovides a method of enzymatic modification of nucleic acids, such assubstrate attached probes, in a capillary gap flow cell. Those skilledin the art will recognize that a capillary flow cell can be formed withany of a variety of arrays known in the art to achieve similar fluidflow capabilities.

The sites can be a pattern such as a regular design or configuration, orthe sites can be in a non-patterned distribution. A non-limitingadvantage of a regular pattern of sites is that the sites can beconveniently addressed in an X-Y coordinate plane. A pattern in thissense includes a repeating unit cell, such as one that allows a highdensity of beads on a substrate.

In a particular embodiment, an array substrate can be an optical fiberbundle or array, as is generally described in U.S. Ser. No. 08/944,850,U.S. Pat. No. 6,200,737; WO9840726, and WO9850782. Also useful in theinvention is a preformed unitary fiber optic array having discreteindividual fiber optic strands that are co-axially disposed and joinedalong their lengths. A distinguishing feature of a preformed unitaryfiber optic array compared to other fiber optic formats is that thefibers are not individually physically manipulable; that is, one strandgenerally cannot be physically separated at any point along its lengthfrom another fiber strand.

The sites of an array of the invention need not be discrete sites. Forexample, it is possible to use a uniform surface of adhesive or chemicalfunctionalities, for example, that allows the attachment of particles atany position. That is, the surface of an array substrate can be modifiedto allow attachment or association of microspheres at individual sites,whether or not those sites are contiguous or non-contiguous with othersites. Thus, the surface of a substrate can be modified to form discretesites such that only a single bead is associated with the site or,alternatively, the surface can be modified such that beads end uprandomly populating sites in various numbers.

In a particular embodiment, the surface of the substrate can be modifiedto contain wells, or depressions in the surface of the substrate. Thiscan be done using a variety of techniques, including, but not limitedto, photolithography, stamping techniques, molding techniques ormicroetching techniques. As will be appreciated by those in the art, thetechnique used will depend on the composition and shape of thesubstrate. When the substrate for a composite array is a microtiterplate, a molding technique can be utilized to form bead wells in thebottom of the assay wells.

In a particular embodiment, physical alterations can be made in asurface of a substrate to produce array locations. For example, when thesubstrate is a fiber optic bundle, the surface of the substrate can be aterminal end of the fiber bundle, as is generally described in U.S. Pat.Nos. 6,023,540 and 6,327,410. In this embodiment, wells can be made in aterminal or distal end of a fiber optic bundle having several individualfibers, In this embodiment, the cores of the individual fibers can beetched, with respect to the cladding, such that small wells ordepressions are formed at one end of the fibers. The depth of the wellscan be altered using different etching conditions to accommodateparticles of a particular size or shape. Generally in this embodiment,the microspheres are non-covalently associated in the wells, althoughthe wells can additionally be chemically functionalized for covalentbinding of particles. As set forth below in further detail,cross-linking agents can be used, or a physical barrier can be used suchas a film or membrane over the particles.

In a particular embodiment, the surface of a substrate can be modifiedto contain chemically modified sites that are useful for attaching,either-covalently or non-covalently, probes or particles having attachedprobes. Chemically modified sites in this context include, but are notlimited to, the addition of a pattern of chemical functional groupsincluding, for example, amino groups, carboxy groups, oxo groups orthiol groups. Such groups can be used to covalently attach probes orparticles that contain corresponding reactive functional groups. Otheruseful surface modifications include, for example, the addition of apattern of adhesive that can be used to bind particles; the addition ofa pattern of charged groups for the electrostatic attachment of probesor particles; the addition of a pattern of chemical functional groupsthat render the sites differentially hydrophobic or hydrophilic, suchthat the addition of similarly, hydrophobic or hydrophilic probes orparticles under suitable conditions will result in association to thesites on the basis of hydroaffinity.

Once microspheres are generated, they can be added to a substrate toform an array. Arrays can be made, for example, by adding a solution orslurry of the beads to a substrate containing attachment sites for thebeads. A carrier solution for the beads can be a pH buffer, aqueoussolvent, organic solvent, or mixture. Following, exposure of a beadslurry to a substrate, the solvent can be evaporated, and excess beadsremoved. In embodiments wherein non-covalent methods are used toassociate beads to an array substrate, beads can be loaded onto thesubstrate by exposing the substrate to a solution of particles and thenapplying energy, for example, by agitating or vibrating the mixture.However, static loading can also be used if desired. Methods for loadingbeads and other particles onto array substrates that can be used in theinvention are described, for example, in U.S. Pat. No. 6,355,431. Beadloading can be carried out prior to modification of probes in adetection method set forth herein. Alternatively, bead loading can becarried out after modification of bead immobilized probes that arehybridized with genome fragments in a method of the invention.

In some embodiments, for example when chemical attachment is done,probes or particles with associated probes can be attached to asubstrate in a non-random or ordered process. For example, usingphotoactivatible attachment linkers or photoactivatible adhesives ormasks, selected sites on an array substrate can be sequentiallyactivated for attachment, such that defined populations of probes orparticles are laid down at defined positions when exposed to theactivated array substrate.

Alternatively, probes or particles with associated probes can berandomly deposited on a substrate and their positions in the arraydetermined by a decoding step. This can be done before, during or afterthe use of the array to detect typable loci using methods such as thoseset forth herein. In embodiments where the placement of probes israndom, a coding or decoding system can be used to localize and/oridentify the probes at each location in the array. This can be done inany of a variety of ways, as is described, for example, in U.S. Pat. No.6,355,431.

In embodiments where particles are used, unique optical signatures canbe incorporated into the particles and can be used to identify thechemical functionality or nucleic acid associated with the particle.Exemplary optical signatures include, without limitation, dyes, usuallychromophores or fluorophores, entrapped or attached to the beads.Different types of dyes, different ratios of mixtures of dyes, ordifferent concentrations of dyes, or a combination of these differencescan be used as optical signatures in the invention. Further examples ofparticles and other supports having detectable signatures that can beused in the invention are described in Currin et al., Nature Materials1:39-41 (2002); U.S. Pat. No. 6,023,540 or 6,327,410; or WO9840726. Inaccordance with this embodiment, the synthesis of the nucleic acids canbe divorced from their placement on an array. Thus, capture probes canbe synthesized on beads, and then the beads can be randomly distributedon a patterned surface. Since the beads are first coded with an opticalsignature, this means that the array can later be decoded. Thus, afteran array is made, a correlation of the location of an individual arraylocation on the array with its probe identity can be made. This meansthat the array locations can be randomly distributed on the array, afast and inexpensive process in many applications of the invention ascompared to either in situ synthesis or spotting techniques that aregenerally outlined in U.S. Ser. Nos. 98/05025, 99/14387, 08/818,199 or09/151,877. However, if desired, arrays made by in situ synthesis orspotting techniques can be used in the invention.

It should be noted that not all sites of an array need to include aprobe or particle. Thus, an array can have one or more array locationson the substrate that are empty. In some embodiments, an array substratecan include one or more sites that contain more than one bead or probe.

As will be appreciated by those in the art, a random array need notnecessarily be decoded. In this embodiment, beads or probes can beattached to an array substrate, and a detection assay performed. Arraylocations that have a positive signal for presence of a probe-fragmenthybrid with a particular typable locus can be marked or otherwiseidentified to distinguish or separate them from other array locations.For example, in applications where beads are labeled with a fluorescentdye, array locations for positive or negative beads can be marked byphotobleaching. Further exemplary marks include, but are not limited to,non-fluorescent precursors that are converted to fluorescent form bylight activation or photocrosslinking groups which can derivatize aprobe or particle with a label or substrate upon irradiation with lightof an appropriate wavelength.

In a particular embodiment, several levels of redundancy can be builtinto an array used in the invention. Building redundancy into an arraycan give several non-limiting advantages, including the ability to makequantitative estimates of confidence about the data and substantialincreases in sensitivity. As will be appreciated by those in the art,there are at least two types of redundancy that can be built into anarray: the use of multiple identical probes or the use of multipleprobes directed to the same target, but having different chemicalfunctionalities. For example, for the detection of nucleic acids, sensorredundancy utilizes a plurality of sensor elements such as beads havingidentical binding ligands such as probes. Target redundancy utilizessensor elements with different probes to the same target: one probe canspan the first 25 bases of a target, a second probe can span the second25 bases of the target, etc. By building in either or both of thesetypes of redundancy into an array a variety of statistical mathematicalanalyses can be done for analysis of large data sets. Other methods fordecoding with redundant sensor elements and target elements that can beused in the invention are described, for example, in U.S. Pat. No.6,355,431.

Typable loci of probe-fragment hybrids can be detected on an array usingthe methods set forth previously herein. In a particular embodiment,probe redundancy can be used. In this embodiment, a plurality of probeshaving identical sequences is present in an array. Thus, a plurality ofsubpopulations each having a plurality of beads with identical probescan be present in the array. By using several identical probes for agiven array, the optical signal from each array location can be combinedand analyzed using statistical methods. Thus, redundancy cansignificantly increase the confidence of the data where desired.

As will be appreciated by those in the art, the number of identicalprobes in a sub-population will vary with the application and use of aparticular array. In general, anywhere from 2 to thousands of identicalarray locations can be used, including, for example, about 5, 10, 20, 50or 100 identical probes or particles.

Once obtained, signals indicative of probe-fragment hybrids from aplurality of array locations can be manipulated and analyzed in avariety of ways, including baseline adjustment, averaging, standarddeviation analysis, distribution and cluster analysis, confidenceinterval analysis, mean testing, or the like. Further description of thedata manipulations is set forth below and in many cases is exemplifiedfor probe-fragment hybrids detected on a bead array. Those skilled inthe art will recognize that similar manipulations can be carried out forother populations of probe-fragment hybrids including, for example,those in which other array locations are treated similarly to the beadsin the examples below.

Optionally, a plurality of signals detected from an array or othermixture of probe-fragment hybrids can be baseline adjusted. In anexemplary procedure, optical signals can be adjusted to start at a valueof 0.0 by subtracting the integer 1.0 from all data points. Doing thisallows the baseline-loop data to remain at zero even when summedtogether and random response signal noise is canceled out. When thesample is a fluid, the fluid pulse-loop temporal region, however,frequently exhibits a characteristic change in response, eitherpositive, negative or neutral, prior to the sample pulse and oftenrequires a baseline adjustment to overcome noise associated with driftin the first few data points due to charge buildup in the CCD camera. Ifno drift is present, typically the baseline from the first data pointfor each bead can be subtracted from all the response data for the samebead type. If drift is observed, the average baseline from the first tendata points for each bead can be subtracted from all the response datafor the same bead type. By applying this baseline adjustment, whenmultiple array location responses are added together they can beamplified while the baseline remains at zero. Since all array locationsrespond at the same time to the sample (e.g. the sample pulse), they allsee the pulse at the exact same time and there is no registering oradjusting needed for overlaying their responses. In addition, othertypes of baseline adjustment that are known in the art can be performed,depending on the requirements and output of the system used.

Any of a variety of possible statistical analyses can be run to generateknown statistical parameters. Analyses based on redundancy are known andgenerally described in texts such as Freund and Walpole, MathematicalStatistics, Prentice Hall Inc., New Jersey (1980).

If desired, signal summing can be done by adding the intensity values ofall responses at a particular time point. In a particular embodiment,signals can be summed at several timepoints, thereby generating atemporal response comprised of the sum of all bead responses. Thesevalues can be baseline-adjusted or raw. Signal summing can be performedin real time or during post-data acquisition data reduction andanalysis. In one embodiment, signal summing can be performed with acommercial spreadsheet program (Excel, Microsoft, Redmond, Wash.) afteroptical response data is collected. Further exemplary signal summingmethods that can be used in the invention are described in U.S. Pat. No.6,355,431.

In a particular embodiment, statistical analyses can be done to evaluatewhether a particular data point has statistical validity within asubpopulation by using techniques including, but not limited to,distribution or cluster analysis. This can be done to statisticallydiscard outliers that can otherwise skew the result and increase thesignal-to-noise ratio of any particular experiment. Useful methods fordetermining whether data points have statistical validity are described,for example, in U.S. Pat. No. 6,355,431 and include, but are not limitedto, the use of confidence intervals, mean testing, or distributionanalysis.

A particular embodiment utilizes a plurality of nucleic acid probes thatare directed to a single typable locus but differ in their actualsequence. For example, a single target genome fragment can have two ormore array locations each having a different probe. This can add a levelof confidence in applications where non-specific binding interactionsoccur with particular sequences. Accordingly, redundant nucleic acidprobes can have sequences that are overlapping, adjacent, or spatiallyseparated.

A method of the invention can further include a step of contacting anarray of nucleic acid probes with chaperone probes. Chaperone probes arenucleic acids that hybridize to a target genome fragment at a site thatis proximal to the hybridization site for a probe used to detect orcapture the genome fragment. Chaperone probes can be added before orduring a capture step or detection step in order to favor hybridizationof capture probes or detection probes to the genome fragment. Chaperoneprobes can favor hybridization of detection or capture probes bypreventing association of the complementary strands of a genome fragmentsuch that the appropriate template strand is available for annealing tothe detection or capture probes.

Chaperone probes can have any of a variety of lengths or compositionsincluding, for example, those set forth previously herein for othernucleic acids useful in the invention. A chaperone probe can hybridizeto a target sequence immediately adjacent to an annealing site foranother probe or at a site that is separated from the annealing site forthe other probe. The gap between probes can be 1 or more, 2 or more, 3or more, 5 or more, 10 or more nucleotides in length or longer.Chaperone probes can be provided in any stoichiometric concentrationthat is found to effectively favor annealing of another probe including,for example, a ratio of about 100 moles, 10 moles, 5 moles, 2 moles, 1mole, 0.5 mole, or 0.1 mole of chaperone probe per mole of target genomefragment.

A method of the invention can further include a step of signalamplification in which the number of detectable labels attached to anucleic acid is increased. In one embodiment, a signal amplificationstep can include providing a nucleic acid that is labeled with a ligandhaving affinity for a particular receptor. A first receptor having oneor more sites capable of binding the ligand can be contacted with thelabeled nucleic acid under conditions where a complex forms between thereceptor and ligand-labeled nucleic acid. Furthermore, the receptor canbe contacted with an amplification reagent that has affinity for thereceptor. The amplification reagent can be, for example, the ligand, amimetic of the ligand, or a second receptor having affinity for thefirst receptor. The amplification reagent can in turn be labeled withthe ligand such that a multimeric complex can form between the ligandreceptor and amplification reagent. The presence of the multimericcomplex can then be detected, for example, by detecting the presence ofa detectable label on the receptor or the amplification reagent. Thecomponents included in a signal amplification step can be added in anyorder so long as a detectable complex is formed. Furthermore, otherbinding moieties and binding partner pairs such as those set forthherein previously can be used for signal amplification.

As shown in the exemplary signal amplification scheme of FIG. 10 ,signal amplification can be carried out using a nucleic acid labeled bystreptavidin-phycoerythrin (SAPE) and a biotinylated anti-SAPE antibody.In one embodiment, a three step protocol can be employed in whicharrayed probes that have been modified to incorporate biotin are firstincubated with streptavidin-phycoerythrin (SAPE), followed by incubationwith a biotinylated anti-streptavidin antibody, and finally incubationwith SAPE again. This process creates a cascading amplification sandwichsince streptavidin has multiple antibody binding sites and the antibodyhas multiple biotins. Those skilled in the art will recognize from theteaching herein that other receptors such as avidin, modified versionsof avidin, or antibodies can be used in an amplification complex andthat different labels can be used such as Cy3, Cy5 or others set forthpreviously herein. Further exemplary signal amplification techniques andcomponents that can be used in the invention are described, for example,in U.S. Pat. No. 6,203,989 B1.

A method of the invention can further include a step of removing genomefragments from probe-fragment hybrids following modification of theprobes and prior to detection of the modified probes, Genome fragmentscan be removed by denaturing fragment-probe hybrids using methods knownin the art for disrupting base-pairing interactions such as exposure tolow salt, organic solvents such as formamide, heat or other denaturingagents. Exemplary methods for denaturing hybrid nucleic acids that areuseful in the methods are described in Sambrook et al., supra (2001) orin Ausubel et al., supra, (1998). Genome fragments can be washed awayfollowing denaturation. Alternatively, genome fragments can be presentunder denaturing conditions during detection.

A method of the invention can further include a step of producing areport identifying at least one typable locus that is detected. Adetected typable locus can be directly identified for example, bysequence, location on a chromosome or by a recognized name of the locus.Alternatively, the report can include data obtained from a method of theinvention in a format that can be subsequently analyzed to identify oneor more detected loci.

Thus, the invention further provides a report of at least one resultobtained by a method of the invention. A report of the invention can bein any of a variety of recognizable formats including, for example, anelectronic transmission, computer readable memory, an output to acomputer graphical user interface, compact disk, magnetic disk or paper.Other formats suitable for communication between humans, machines orboth can be used for a report of the invention.

The invention further provides an array including a solid-phaseimmobilized representative population of genome fragments. Arepresentative population of genome fragments can be produced andimmobilized using methods such as those set forth herein previously, Forexample, a genome can be amplified using primers having a secondarylabel such as biotin or reactive crosslinking groups and subsequentlyimmobilized via interaction with a solid phase receptor such as avidinor a chemical moiety reactive with the crosslinking group. A solid-phaseimmobilized representative population of genome fragments can have oneor more of the characteristics set forth previously herein such as high,low or medium complexity.

A solid-phase immobilized representative population of genome fragmentscan be directly interrogated using the methods of the invention.Generally, detection assays and methods have been exemplified above withrespect to immobilized probes and soluble genome fragment targets. Thoseskilled in the art will recognize that in embodiments wherein arepresentative population of genome fragments is immobilized the methodscan be similarly performed, however, with the genome fragments replacingthe probes in the above examples and the probes treated as targets inthe above examples.

Employing a solid phase genomic DNA target can provide the advantage ofa high degree of assay multiplexing by allowing any poorly hybridized orexcess detection primers to be washed away before subsequent enzymaticmodification of the primers, for example, in an extension or ligationtechnique. Applications that are adversely affected by primer-dimerformation can be improved by removing primer dimers before detection. Asolid-phase target DNA format can also allow fast hybridization kineticssince the primers can be hybridized at a relatively high concentrations,for example, greater than about 100 pM.

The methods set forth herein for amplifying genomic DNA allow relativelysmall amounts of genomic DNA to be amplified to a large amount.Immobilization of large amounts of genomic DNA to a solid-phase canallow typable loci to be queried directly, for example, in a primerextension or ligation-based assay without the need for subsequentamplification, Elimination of amplification can lead to more robust andquantitative genotyping than is often available whenpre-amplification-based detection is used.

Another advantage of using a solid phase genomic DNA target is that itcan be reused. Thus, the immobilized genome target can be an archivalsample that can be used repeatedly with different sets of nucleic acidprobes. Furthermore, in some applications carry-over contamination canbe reduced by using immobilized gDNA since the amplification occursbefore the SNP specific detection reaction. It will be understood that,the steps described above for carrying out methods of the invention havebeen set forth in a particular order for the sake of explanation. Thoseskilled in the art will recognize that the steps can be carried out inany of a variety of orders so long as a desired result is achieved. Forexample, components of the reactions set forth above can be addedsimultaneously, or sequentially, in any order that are effective atproducing one or more of the results described, In addition, thereactions set forth herein can include a variety of other reagentsincluding, for example, salts, buffers, neutral proteins, albumin,detergents, or the like. Such reagents can be added to facilitateoptimal hybridization and detection, reduce non-specific or backgroundinteractions, or to stabilize other reagents used. Also reagents thatotherwise improve the efficiency of a method of the invention, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, or thelike can be used, depending on the sample preparation methods and purityof the target. Those skilled in the art will know or be able todetermine appropriate reagents to achieve such results.

Several of the methods exemplified herein with respect to detection oftypable loci of genomic DNA can also be applied to gene expressionanalysis. In particular, methods for on-array labeling of probe nucleicacids using primer extension methods can be used in the detection of RNAor cDNA. Probe-cDNA hybrids can be detected by polymerase-based primerextension methods as described herein previously. Alternatively, forarray-hybridized mRNA, reverse-transcriptase-based primer extension canbe employed. There are several non-limiting advantages of on-arraylabeling for gene expression analysis, Labeling costs can bedramatically decreased since the amounts of labeled nucleotides employedare substantially less compared to methods for labeling capturedtargets. Secondly, cross-hybridization can be dramatically reduced sincea target must both hybridize and also contain perfect complementarity atits 3′ terminus for label incorporation in a primer extension reaction.Similarly, GLA or GoldenGate™ assays can be used for detection ofhybridized cDNA or mRNA. The latter two methods typically requireaddition of an exogenous nucleic acid for each locus queried. However,such methods can be advantageous in applications where the use of primerextension leads to unacceptable levels of ectopic extension.

The above described on-array labeling with primer extension can also beused to monitor alternate splice sites by designing the 3′ probeterminus to coincide with a splice junction of a target cDNA or mRNA.The terminus can be placed to uniquely identify all the relevantpossible acceptor splice sites for a particular gene. For example, thefirst 45 bases can be chosen to lie entirely within the donor exon, andthe last 5 3′-bases can lie in a set of possible splice acceptor exonsthat become spliced adjacent to the first 45 bases.

A cDNA or mRNA target can be used in place of gDNA in a method describedpreviously herein for identifying typable loci. For example, a cDNA ormRNA target can be used in a genotyping assay. Genotyping cDNA or mRNAcan allow allelic-specific expression differences to be monitored, forexample, via “quantitative genotyping”, or measuring the proportion ofone allele vs. the other allelic at a biallelic SNP marker. Allelicexpression differences can result, for example, from changes intranscription rate, transcript processing or transcript stability. Suchan effect can result from a polymorphism (or mutation) in a regulatoryregion, promoter, splice site or splice site modifier region or othersuch regions. In addition, epigenomic changes in the chromatin such asmethylation can also contribute to allelic expression differences. Thus,the methods can be used to detect such polymorphisms or mutations inexpressed products.

A “normalized” representation can be created from a cDNA or mRNA targetby any of several methods such as those based upon placing universal PCRtails on a cDNA representation (see, for example, Brady, Yeast, 17:211-7(2000)) The normalization process can be used to generate a cDNArepresentation wherein each typable locus in the population is presentat relatively the same copy number. This can aid in the quantitativegenotyping process of a cDNA sample since the signal intensities fromthe array-based primer extension assay will be more uniform than withoutthe normalization process.

In a further embodiment, a method of the invention can be used tocharacterize an mRNA or cDNA sample. An mRNA or cDNA sample can be usedas a target sample in a method of the invention and a representative setof typable loci detected. The representative set of typable loci can beselected to be diagnostic or characteristics of the mRNA or cDNA sample.For example, the levels of particular typable loci can be detected in asample and compared to reference levels for these loci, the referencelevels being indicative of the extent to which the sample includesexpressed sequences covering desired genes. Thus, the methods can beused to determine the quality of an mRNA or cDNA sample or itsappropriateness for a particular application.

A typical array location, such as a bead, can contain a large populationof relatively densely packed probe nucleic acids. Followinghybridization of target nucleic acids under many conditions only aportion of probes in a detection assay will be occupied with acomplementary target. Under such conditions it is possible that denselypacked probes will form inter-probe structures that are susceptible toectopic primer extension. Furthermore, as shown in FIG. 13A probeshaving self-complementary sequences can also structures that aresusceptible to ectopic primer extension. Ectopic extension refers tomodification of one or both probes in an inter- or intra-probe hybridduring an extension reaction. Ectopic extension can occur regardless ofthe presence of a hybridized target to the array.

Accordingly, the invention provides a method for inhibiting ectopicextension of probes in a primer extension assay. The method includes thesteps of (a) contacting a plurality of probe nucleic acids with aplurality of target nucleic acids under conditions wherein probe-targethybrids are formed; (b) contacting the plurality of probe nucleic acidswith an ectopic extension inhibitor under conditions whereinprobe-ectopic extension inhibitor hybrids are formed; and (c)selectively modifying probes in the probe-target hybrids compared toprobes in the probe-ectopic extension inhibitor hybrids.

An ectopic extension inhibitor useful in the invention can be any agentthat is capable of binding to a single stranded nucleic acid probe,thereby preventing hybridization of the probe to a second probe.Exemplary agents include, but are not limited to single stranded nucleicacid binding proteins (SSBs), nucleic acids such as those set forthabove including nucleic acid analogs, small molecules. Such agents havethe general property of preferentially binding to single-strandednucleic acids over double-stranded nucleic acids irrespective of thenucleotide sequence. Exemplary single-stranded nucleic acid bindingproteins that can be used in the invention include, but are not limitedto, Eco SSB, T4 gp32, T7 SSE3, N4 SSB, Ad SSB, UP1, and the like andothers described, for example, in Chase et al, Ann. Rev. Biochem., 55:103-36 (1986); Coleman et al, CRC Critical Reviews in Biochemistry,7(3): 247-289 (1980) and U.S. Pat. No. 5,773,257. Ectopic extension inany of the primer extension assays set forth above can be inhibitedusing a method of the invention. Exemplary embodiments of the methodsfor inhibiting ectopic extension of probes in a primer extension assayare shown in FIG. 13 and described in further detail below.

As shown in FIG. 13B, ectopic extension can be minimized by incubating apopulation of probes with a protein or other agent that selectivelybinds single stranded nucleic acids, such as SSB, T4 gene 32 or thelike. The agent or protein can be added under conditions where it coatsthe single strand probes that have not hybridized to a target nucleicacid thereby preventing their self-annealing and subsequent extension.An agent such as a protein that binds to single stranded probes can beadded to a. population of probes prior to or during a primer extensionreaction, for example, prior to or during an annealing step.

Ectopic expression can also be reduced using one or more blockingoligonucleotides (oligos). As shown in FIG. 13C, a blocking oligo thatis complementary to the 3′ end of a probe can be added under conditionswhere it will hybridize to probes that have not hybridized to a targetnucleic acid. In applications where several probes are present, aplurality of blocking oligonucleotides designed to anneal to the 3′ endsof the probes can be added. One or more blocking oligos can be added toa population of probes prior to or during a primer extension reaction,for example, prior to or during an annealing step.

As shown in FIG. 13D, a probe can be designed with complementarysequence portions capable of forming a hairpin structure that is notcapable of being extended under the conditions used for the primerextension step in a primer extension assay. In the example shown in FIG.13D, the 3′ end of the probe anneals to the 5′ end of the probe, andbecause the 5′ end is not adjacent to a readable template the hairpincannot be ectopically extended. A probe can be designed to have a firstsequence region adjacent to the 3′ end of the probe that iscomplementary to a second sequence region of the probe such that ahairpin forms with a 3′ overhang that is not capable of being extended.The hairpin structure is further designed such that it does not inhibitannealing to target nucleic acids under conditions of the annealing stepof a primer extension reaction. For example, two regions of a probe canhave complementary sequences that do not substantially anneal attemperatures used during target hybridization, but become annealed toform a hairpin once the temperature is reduced for extension.

Although methods for reducing ectopic extension are exemplified abovewith respect to arrayed probes, those skilled in the art will recognizethat the methods can be similarly applied to extension reactions inother formats such as solution phase reactions or beads spatiallyseparated in fluid phase.

Under some extension assay conditions polymerases can place extranucleotides at the end of 3′ termini of a single stranded probe absent ahybridized template nucleic acid. Such an activity is also known tooccur at the 3′ termini of blunt ends of double stranded nucleic acidsunder some conditions and is referred to as a terminal extendaseactivity (see for example, Hu et al., DNA and Cell Biology, 12:763-770(1993). Accordingly, an extension reaction used in the invention can becarried out under conditions that inhibit terminal extendase activity.For example, a polymerase can be selected that has sufficiently lowlevels of terminal extendase activity under the extension reactionconditions to be used or nucleotides that are preferentiallyincorporated by the extendase activity of a particular polymerase can beexcluded from an extension reaction, or unhybridized probes can beblocked or removed from an extension reaction.

Direct hybridization detection of nucleic acid targets can suffer fromdecrease the assay specificity due to cross-hybridization reactionsunder some assay conditions. Array-based enzymatic detection of nucleicacid targets offers a powerful approach to increase specificity. Inaddition to the field of genotyping previously discussed, the inventioncan be applied to increasing specificity in detection of DNA copynumber, microbial agents, gene expression, and so forth. This becomesparticularly relevant as the complexity of the nucleic acid sampleincreases to the level of human genomic complexity. For instance, DNAcopy number experiments in which labeled genomic DNA is hybridized toDNA arrays are often compromised by specificity problems. By employingdirect hybridization in combination with an array-based enzymatic stepsuch as primer extension, or others set forth previously herein,specificity can be dramatically improved. This is becausecross-hybridizing targets will not be detected since labeling by theenzymatic detection step occurs due to perfect 3′ complementarily.

In accordance with another embodiment of the present invention, thereare provided diagnostic systems for carrying out one or more of themethods described previously herein. A diagnostic system of theinvention can be provided in kit form including, if desired, a suitablepackaging material. In one embodiment, for example, a diagnostic systemcan include a plurality of nucleic acid probes, for example, in an arrayformat, and one or more reagents useful for detecting a gDNA fragment orother target nucleic acid hybridized to a probe of the array.Accordingly, any combination of reagents or components that is useful ina method of the invention, such as those set forth herein previously inregard to particular methods, can be included in a kit provided by theinvention. For example, a kit can include one or more nucleic acidprobes bound to an array and having free 3′ ends along with otherreagents useful for a primer extension detection reaction.

As used herein, the phrase “packaging material” refers to one or morephysical structures used to house the contents of the kit, such asnucleic acid probes or primers, or the like. The packaging material canbe constructed by well-known methods, preferably to provide a sterile,contaminant-free environment. The packaging materials employed hereincan include, for example, those customarily utilized in nucleicacid-based diagnostic systems. Exemplary packaging materials include,without limitation, glass, plastic, paper, foil, and the like, capableof holding within fixed limits a component useful in the methods of theinvention such as an isolated nucleic acid, oligonucleotide, or primer.

The packaging material can include a label which indicates that theinvention nucleic acids can be used for a particular method. Forexample, a label can indicate that the kit is useful for detecting aparticular set of typable loci, thereby determining an individual'sgenotype. In another example, a label can indicate that the kit isuseful for amplifying a particular genomic DNA sample.

Instructions for use of the packaged reagents or components are alsotypically included in a kit of the invention. “Instructions for use”typically include a tangible expression describing the reagent orcomponent concentration or at least one assay method parameter, such asthe relative amounts of kit components and sample to be admixed,maintenance time periods for reagent/sample admixtures, temperature,buffer conditions, and the like.

A method of the invention can include controls for determining desirableor undesirable outcome for one or more of the reagents, components, orsteps disclosed herein. Comparison of results for a sample beinginvestigated with results for controls can be performed in a method ofthe invention, thereby validating results, identifying steps that bearrepeating or influencing interpretation of results. If the results forone or more controls are outside of a desired range of results a method,of the invention can include a step of modifying a value or other datapoint obtained for a sample being investigated. A method of theinvention can include determining results for one or more of thecontrols set forth below and if the results are outside of a desiredrange then repeating one or more steps of the method. Thus, detection ofa signal from a control and modification of conditions can be carriedout in an iterative fashion until a desired set of condition isobtained.

Amplification controls can be used in a method of the invention such asa method including a step of representationally amplifying a genomeand/or producing genome fragments. An exemplary amplification control isan extrinsic genome spike. For example, a small amount of microbialgenomic DNA can be spiked into a reaction for random primeramplification of a human genome. The amount of microbial genomic DNAadded is typically sufficient to compete with potential contaminationfrom other DNA samples but insufficient to substantially compete withamplification of the human genomic DNA sample, Detection of loci thatare unique to the microbial genome compared to the human genome using,for example, a subset of probes that selectively hybridize to themicrobial loci and not to human loci, can be used to determine whether afailed amplification is due to faulty RPA reaction components or poorquality human genomic DNA. More specifically, detectable levels ofmicrobial loci resulting from the RPA reaction indicate that the humangenomic DNA is poor quality and RPA reaction components are functionaland, in contrast, absence of detectable levels of microbial lociindicate a failure of the reaction components.

Hybridization controls can be used in a method of the invention such asa method including a step of contacting genome fragments with a nucleicacid probes. Typically, hybridization controls are synthetic nucleicacids that are co-incubated with targets nucleic acids during a probehybridization step. An example of a useful hybridization control is aset of stringency control probes having sequences forming a series ofmismatches relative to the sequence of a stringency control target. Theprobe series can include a first probe having a sequence that is aperfect match with the sequence of the stringency control target, asecond probe having a mismatch with the sequence of the stringencycontrol target, a third probe having the same mismatch as the secondprobe and a second mismatch, a fourth probe having the same twomismatches as the third probe and a third mismatch etc. It is possibleto have two or more mismatches per probe in this series. The mismatchesin the series can be adjacent to each other or spaced apart from eachother such that one or more matching nucleotides intervenes in thesequence. The mismatches in the series can be located near the 5′ end ofthe probe such that all of the probes have a 3′ end that matchesperfectly with the stringency control target.

The number and/or identity of the stringency control probes thathybridize to the stringency control target can be correlated with thestringency of the hybridization conditions. At the highest stringencylevels only the first stringency control probe in the above series (theperfect match control probe) will hybridize to the target control probe.Lower stringency conditions will result in more of the stringencycontrol probes in the series hybridizing to the stringency controltarget. Thus, stringency control probes can be used to identifyconditions that provide a desired stringency for hybridization forgenome fragments and probes.

A further control that can be used is a concentration control. Aconcentration control target having a sequence that is a perfect matchwith the sequence of a concentration control probe can be used.Concentration control targets can be provided at differentconcentrations to control probes. The lower limit of detection for aparticular set of assay conditions can be quantified by determining thelowest concentration of target detected. If desired, the concentrationcontrol target can have one or more mismatches to the control probe, forexample, at the 3′ end of the target sequence. Accordingly, stringencyor specificity evaluation can also be made with concentration probes.

Probe modification controls can be used in a method of the inventionsuch as a method including a step of modifying a probe while hybridizedto a genome fragment. Examples of probe modification controls areextension controls that indicate levels of probe extension by polymerasein a method of the invention. An exemplary extension control is ahairpin probe or set of match and mismatch. hairpin probes. The set caninclude two or more of the 16 possible combinations of matches andmismatches that arise for 4 nucleotides (for example, a GC match and atleast one of GA, GT and GG). Hairpin probes are typically attached to asubstrate at their 5′ ends and have a palindromic sequence such thatthey can form a hairpin structure at their 3′ ends under permissiblestringency conditions. The match probe will have a hairpin terminatingin a 3′ base pair match, whereas the mismatch probe will terminate in a3′ mismatch. Modification of the match hairpin probe indicates that theextension assay components are functional under the conditions beingemployed. An advantage of using hairpin control probes is that theindication is independent of presence of target nucleic acids. Thus, fora failed extension reaction the results for the match hairpin controlcan be used to determine if problems arose from the target nucleic acidsample or the other extension reaction reagents. Modification of themismatch hairpin probe can be monitored to determine if the extensionreaction reagents are modifying probes in a template independentfashion. Although the hairpin control probes have been exemplified abovewith respect to extension reactions, those skilled in the art willrecognize that they can be used in other template-dependent modificationreactions such as a ligation reaction.

Another useful probe modification control is an extension efficiencycontrol. An extension efficiency control can include a set of extensionefficiency control probes that are complementary to overlappingsequences of an extension efficiency control target such that the 3′ends of the probes complement an A, C, T or G nucleotide, respectively,of a 4 nucleotide sequence. Thus, a sequence alignment of an extensionefficiency control target with four such extension efficiency controlprobes appears as a staggered set of sequences offset at their 3′ endsby one nucleotide. An extension efficiency control can be useful fordetermining whether or not selected extension reaction conditions arebalanced with respect to incorporating all of the nucleotides being usedor if one or more nucleotide is being incorporated selectively,

A method of the invention can further include evaluation ofnon-polymorphic controls. A non-polymorphic control is a set of perfectmatch and mismatch probes for a non-polymorphic sequence in a genome.The perfect match and mismatch probes are complementary to the sameregion of the genome with the exception that their 3′ ends are eithercomplementary or non-complementary, respectively, to the genome sequenceregion. One or more sets can be used, for example, having different GCcontents to monitor stringency, and/or having one or more of allpossible combinations of matches and mismatches. Polymorphic probes canfacilitate assay optimization using single or mixed individual sampleswhen compared to clustering data with multiple individuals.

Strip controls can be used in a method of the invention such as a methodincluding a step of removing genome fragments from a plurality ofprobes, For example, a labeled strip control target can be spiked into agenome fragment sample prior to hybridization with a plurality of probessuch that once the hybrids have been treated to remove genome fragmentsthe presence or absence of the labeled target can be detected andcorrelated with unsatisfactory or satisfactory fragment removal,respectively. In particular embodiments, a label can be incorporatedinto a strip control target while hybridized to a complementary probe.For example, the 3′ end of the strip control target can hybridize to theprobe such that the target can be modified in a template dependentfashion. Typically, the strip control target and its complementary probeare designed such that the probe is not modified in the same step as thetarget. For example, the probe can have a 3′ nucleotide analog that isnot amenable to modification and/or the 3′ end of the probe can form amismatch with the target. Furthermore, the probe that complements thestrip control target can be designed to have a sequence that will notcomplement any of the genome fragments to be detected in a method of theinvention,

Detection controls can be used in a method of the invention such as amethod including a step of detecting typable loci of probe-fragmenthybrids. For example, a set of label control probes can be used thathave known amounts of label associated. The label control probes can beanalyzed as a titration curve to determine the sensitivity or range ofdetection for the label used to detect typable loci of genome fragments.The label control probes need not be the same type of molecule as theprobes used for detection of genome fragments. Accordingly, labelcontrol probes can be labels attached directly or indirectly to aparticular location on an array surface. In the case of on-arraybiotin-based detection, label control probes can be array locationshaving known amounts of covalently attached biotin.

Although the invention is exemplified herein with respect to an array ofimmobilized probes, those skilled in the art will recognize that otherdetection formats can be employed as well. For example, the methods setforth herein can be carried out in solution phase rather than solidphase. Accordingly, solution phase probes can replace immobilized probesin the methods set forth above. Solution phase probes can be detectedaccording to properties such as those set forth above in regard todetection labels or detection moieties. For example, probes can haveidentifiable charge, mass, charge to mass ratio or other distinguishingproperties. Such distinguishing properties can be detected, for example,in a chromatography system such as capillary electrophoresis, acrylamidegel, agarose gel or the like, or in a spectroscopic system such as massspectroscopy. Thus, the invention further provides a method of detectingtypable loci of a genome including the steps of (a) providing anamplified representative population of genome fragments having thetypable loci; (b) contacting the genome fragments with a plurality ofnucleic acid probes having sequences corresponding to the typable lociunder conditions wherein probe-fragment hybrids are formed; (c)modifying the probe-fragment hybrids; and (d) detecting a probe orfragment that has been modified, thereby detecting the typable loci ofthe genome.

EXAMPLE I Whole Genome Amplification Using Random-Primed Amplification(RPA)

This example demonstrates production of an amplified representativepopulation of genome fragments from a yeast genome.

Yeast genomic DNA, from S. Cerevisiae strain S228C, was prepared using aQiagen Genomic DNA extraction kit and 10 ng of the genomic DNA wasamplified with Klenow polymerase.

Several parameters were evaluated to determine their effect on the yieldof the Klenow exo) random-primed amplification reaction. Amplificationreactions were carried out under similar conditions with the exceptionthat one parameter was systematically modified. FIG. 3 shows resultscomparing amplification reactions carried out at differentconcentrations of deoxynucleotide triphosphates.

Following each reaction, the amplified DNA was purified on Montageultrafiltration plates (Millipore), loaded onto an agarose gel and theDNA quantitated by UV₂₆₀ reading as shown in FIG. 3A. The amplificationyield was determined based on the density of stain in each lane and theresults are shown in the table in FIG. 3(B). As shown in the last twocolumns of FIG. 3B, 10 ng of yeast genome template was amplified toquantities in the range of about 6 to 80 microgram, representing about600 to 8000 fold amplification. The average fragment size under theconditions tested was about 200-300 bp.

The results demonstrated that amplification yields were increased athigher concentrations of primer or deoxynucleotide triphosphates. Thus,reaction parameters can be systematically modified and evaluated todetermine desired amplification yields.

EXAMPLE II Detection of Yeast Loci for a Yeast Whole Genome SampleHybridized to BeadArrays'

This example demonstrates reproducible detection of yeast loci for ayeast whole genome sample hybridized to a BeadArrays™ and probed withallele-specific primer extension (ASPS).

Six hundred nanograms of random primer amplified (RPA) yeast gDNA washybridized to a locus-specific BeadArray™ (Illumina). The BeadArray™ wascomposed of 96 oligonucleotide probe pairs (PM and MM, 50 bases inlength) interrogating different gene-based loci distributed throughoutthe S. cerevisiae genome. The amplified yeast genomic DNA was hybridizedto the BeadArray™ under the following conditions: Overnighthybridization at 48° C. in standard 1× hybridization buffer (1 M NaCl,100 mM potassium-phosphate buffer (pH 7.5), 0.1% Tween 20, 20%formamide). After hybridization, arrays were washed in 1× hybridizationbuffer at 48° C. for 5 min. followed by a wash in 0.1× hybridizationbuffer at room temperature for 5 min. Finally, the array was washed for5 min, with ASPE reaction buffer to block and equilibrate the arraybefore the extension step. ASPE reaction buffer (10×GG Extension buffer(Illumina, Inc., San Diego, Calif.), 0.1% Tween-20, 100 ug/ml BSA, and 1mM dithiothreitol, 10% sucrose, 500 mM betaine).

An ASPE reaction was performed directly on the array as follows. TheBeadArrays were dipped into 50 uls of an ASPE reaction mix containingthe described ASPE reaction buffer supplemented with 3 uM dNTPs (1.5 uMdCTP), 1.5 uM biotin-11-dCTP, .about.0.4 ul Klentaq (DNA PolymeraseTechnology, Inc, St. Louis, Mo., 63104), The BeadArrays™ were incubatedin the ASPE reaction for 15 min. at room temperature. The BeadArrays™were washed in fresh 0.2 N NaOH for 2 min., then twice in 1×hybridization buffer for 30 sec. The incorporated biotin label wasdetected by a sandwich assay employing streptavidin-phycoerythrin andbiotinylated anti-streptavidin staining. This was done as follows:BeadArrays™ were blocked at room temperature for 30 min in casein block(Pierce, Rockford, Ill.). This was followed by a quick wash (1 min.) in1× hybridization buffer, before staining for 5 min. at room temp. withstreptavidin-phycoerythrin (SAPE) solution (1× hybridization buffer,0.1% Tween 20, 1 mg/ml. BSA, 3 ug/ml streptavidin-phycoetythrin(Molecular Probes, Eugene, Oreg.). After staining, the BeadArrays™ werequick washed with 1×Hyb, buffer before counterstaining with 10 ug/mlbiotinylated anti-streptavidin antibody (Vector Labs, Burlingame,Calif.) in 1×TBS supplemented with 6 mg/ml goat serum, Casein and 0.1%Tween 20. This step was followed by a quick wash in 1×Hyb, buffer, andthan a second staining with SAVE solution as described. After staining,a final wash in 1×Hyb, buffer was performed.

The left panel of FIG. 4 shows an image of an array followinghybridization with amplified whole yeast genome sample and ASPEdetection. The chart in the right panel of FIG. 4 displays a subset ofperfect match (PM) and mismatch (MM) intensities (48 loci out of 96).Greater than 88% of the loci had PM/MM ratios greater than 5 indicatingthe ability to distinguish most loci from alternate genotypes.

The ability to distinguish typable loci in genomes of higher complexitythan yeast was assessed by spiking yeast genomic DNA into the genomicbackground of a more complex organism. Six hundred nanograms Yeastgenomic DNA. (12 Mb complexity) was spiked into 150 ug human genomic DNA(3000 Mb complexity) to mimic the presence of single copy loci in agenome having complexity equivalent to human. Hybridization of thisspiked sample to the array showed very little difference with yeast DNAhybridized alone indicating the ability of the array to specificallycapture the correct target sequences in a complex genomic background.

These results demonstrate detection of several typable loci of a yeastgenome following hybridization of a whole genome sample to an array.These results further demonstrate that amplification is not necessary todetect a plurality of typable loci in a whole genome sample. Furthermorethe results were reproducible showing that the method is robust.

EXAMPLE III Whole Genome Genotyping (WGG) of Human gDNA DirectlyHybridized to Bead Arrays™

This example demonstrates hybridization of a representative populationof genome fragments to an array and direct detection of several typableloci of the hybridized genome fragments, This example furtherdemonstrates detection of typable loci on an array using either of twodifferent primer extension assays.

SBE-Based Detection

Human placental genomic DNA samples were obtained from Corlell Inst.Camden, N.J. The human placental gDNA sample (150 ug) was hybridized toa BeadArray™ (Illumina) having 4 separate bundles each containing thesame set of 24 different non-polymorphic probes (50-mers). TheBeadArray™ consisted of 96 probes to human non-polymorphic loci randomlydistributed throughout the human genome. The probes were 50 bases longwith .about.50% GC content and designed to resequence adjacent A (16probes), C (16 probes), G (16 probes), or T (16 probes) bases. DNAsamples (150 ug human placental DNA) were hybridized overnight at 48° C.in standard 1× hybridization buffer (1 M NaCl, 100 mMpotassium-phosphate buffer (pH 7.5), 0.1% Tween 20, 20% formamide) in avolume of 15 ul.

Four separate SBE reactions were performed directly on the array, onefor each separate bundle, as follows. The “A” reaction containedbiotin-labeled ddATP and unlabeled ddCTP, ddGTP, and ddTTP. The otherthree SBE reactions were similar except that the labeled and unlabeleddesignations were adjusted appropriately. The SBE reaction conditionswere as follows: The BeadArrays™ were dipped into an SBE reaction mix at50° C. for 1 min. Four different SBE reaction mixes were provided, an A,C, G, or T resequencing mix. For example, a 50 ul A-SBE resequencing mixcontained 2 uM biotion-11-ddATP (Perkin Elmer), 1 uM ddCTP, 1 uM ddGTP,and 1 uM ddUTP, 1× Thermosequenase buffer, 0.3 U Thermosequenase, 10ug/ml BSA, 1 mM DTT, and 0.1% Tween 20. The other three SBE mixes weresimilar with the appropriate labeled base included and the other basesunlabeled.

The results of the SBE reactions are shown in FIG. 5 . In FIG. 5 , theset of 96 probes are divided into four groups corresponding to the fourdifferent reactions designated as CA1 through CA24 for thebiotin-labeled ddATP reaction, CC1 through CC24 for the biotin-labeledddCTP reaction, CG1 through CC24 for the biotin-labeled ddGTP reaction,and CT1 through CT24 for the biotin-labeled ddTTP reaction. As shown inFIG. 5 most probes showed excellent signal discrimination,

ASPE-Based Detection

A similarly prepared human placental gDNA sample (150 ug) was hybridizedto a BeadArray™ containing 77 functional perfect match (PM) and mismatch(MM) probe pairs querying non-polymorphic loci. The ASPE probes weredesigned to non-polymorphic sites within the human genome. The probeswere 50 bases in length with .about.50% GC content, The perfect match(PM) probes were completely matched to genomic sequence whereas themismatch (MM) probes contained a single base mismatch to the genomicsequence at the 3′ base. The mismatch type was biased towards modelingA/G and CIT polymorphisms. The hybridization and reaction conditionswere as previously described in Example II.

An allele-specific primer extension reaction (ASPE) was performeddirectly on the array surface, and the incorporated biotin labeldetected with streptavidin-phycoerythrin staining. The ASPE reaction wasperformed as follows. BeadArrays™ were washed twice in 1× hybridizationbuffer and then washed with ASPE reaction buffer (without enzyme andnucleotides) at room temperature. The ASPE reaction was carried out bydipping the BeadArrays™ into a 50 ul A.SPE reaction mix at roomtemperature fur 15 minutes. The ASPE mix contained the followingcomponents: 3 uM dATP, 1.5 uM dCTP, 1.5 uM biotin-11-dCTP, 3 uM dGTP, 3uM dUTP, 1× GoldenGate™ extension buffer (Illumina), 10% sucrose, 500 mMbetaine, 1 mM DTT, 100 ug/ml BSA, 0.1% Tween 20 and 0.4 ul Klentaq (DNAPolymerase Inc., St. Louis, Mo.). FIG. 6A shows the raw intensity valuesacross the 77 probe pairs. The PM probes (squares) exhibit much higherintensities than the MM probes across a majority of the probeseffectively allowing the queried base to be distinguished. FIG. 6B showsa plot of the discrimination ratios (PM/PM+MM) for the 77 loci. Theseresults demonstrated that about two thirds of the loci had ratios >0.8.

The results of this example demonstrate that hybridization of arepresentative population of genome fragments to an array and directdetection of several typable loci of the hybridized genome fragmentsprovides sufficient locus discrimination for genotyping applications.

EXAMPLE IV Genotyping of Amplified Genomic DNA Fragments

This example demonstrates genotyping of an amplified population ofgenome fragments.

Human placental genomic DNA samples were obtained from Coriell Inst.Camden, N.J. The genome was amplified and biotin labeled using randomprimer amplification under conditions described in Example 1, with theexception that the amount of template genome was varied and length ofthe random primer was varied as indicated in FIG. 7 . The amplificationoutput for all reactions was relatively constant at about 40 ug ofamplified genome fragments per 40 ul reaction.

The amplified population of genome fragments was genotyped as follows.The genotyping was performed by Illumina's SNP genotyping services usingthe proprietary GoldenGate™ assay on IllumiCode™ arrays. The GenTrainscore is a metric for how well the genotype intensities of the SNP locicluster across a sample population. A comparison of GenTrain score tothe unamplified control provides an estimate of locus amplification andbias.

The genotyping quality for unamplified. DNA was compared to theamplified population of genome fragments as shown in FIG. 7 . The amountof genome template used in the amplification reaction is shown beloweach bar. Of the amplified samples, the best GenTrain scores wereobtained for the amplification reaction using 1000 ng of template genome(40× amplification). The GenTrain scores for the amplification reactionusing 1000 ng of template genome were similar to that obtained forunamplified genomic DNA, indicating that the amplified product wasrepresentative of the genome. Acceptable GenTrain scores were alsoobtained for amplification reaction using as little as 100 ng oftemplate genome (400× amplification).

These results demonstrate that amplified populations of genome fragmentsobtained in accordance with the invention are representative of thegenome sequence in a genotyping assay.

EXAMPLE V Whole Genome Genotyping (WGG) of Amplified Genomic DNAFragments

This example demonstrates whole genome genotyping of an amplifiedpopulation of genome fragments by direct hybridization to a DNA arrayand array-based primer extension SNP scoring.

A set of 3×32 DNA samples (1 ug each) were amplified by random primeramplification to produce separate target samples having 150 ug ofgenomic DNA fragments. The amplified populations of fragments werehybridized to BeadArrays™ having 50-mer ASPE capture probes covering 192loci. After hybridization, an ASPE reaction was performed as describedin Example III. Images were collected and genotype clusters analyzedusing proprietary GenTrain software (Illumina). An exemplary image of aBeadArray™ detected with ASPE is shown in FIG. 11A.

FIG. 11B shows a GenTrain plot of theta vs. intensity for one locus.Intensity is the total fluorescence intensity detected for a particularbead. Theta corresponds to the position of a bead's fluorescenceintensity on a scatter plot of fluorescence intensity for one allele ofa locus vs. fluorescence intensity for a second allele of the locus. Inparticular, the position of a bead's fluorescence intensity on thescatter plot corresponds to a particular x,y coordinate and theta is theangle between the x axis and a line drawn from the origin to that x,ycoordinate. As shown in FIG. 11B, two homozygous (B/B and A/A) clustersand one heterozygous (A/B) cluster were clearly differentiated.

About 52% of the loci gave well resolved clusters which were termed“successful” loci and were subsequently analyzed for genotypes acrossall the samples. Analysis of the genotype calls (101/192 loci) across3×16 samples for which reference genotypes were known indicated 99.95%concordance (4090/4092) with a call rate of 100% (FIG. 12 , Panel A).GenCall plots showing the scores at different loci are shown in FIGS.12B and C for two different samples. The GenCall score for an individualgenotype call is a value between 0 and 1 that indicates the confidencein that call. A higher score indicates a higher confidence in the call.

Exemplary GenTrain plots for two different loci are shown in FIGS. 12Cand 12D. This data shows that for the majority of samples, threeclusters were clearly differentiated corresponding to homozygous (B/Band A/A) and (A/B) genotypes. The two grey points are from “no targetcontrol” BeadArrays™.

Examination of the scatter plots in FIGS. 12 D and E showed only twoquestionable calls out of 4092 calls, indicated by arrows in the plots.The calls were filtered by applying a threshold of 0.45 for the GenCallscore, as shown by the horizontal line in FIGS. 12B and C.

EXAMPLE VI Inhibition of Ectopic Signals

This example demonstrates the use of single stranded nucleic acidbinding protein (SSB) to inhibit ectopic expression in an array-basedprimer extension reaction.

Single stranded binding proteins such as E. coli SSB and T4 Gene 32 weretested for their ability to suppress ectopic extension in both Mellowand Klentaq array-based ASPE reactions. The conditions employed were asfollows: Array-based Klenow ASPE reaction contained 80 mM Tris-Acetate(pH 6.4), 0.4 mM EDTA, 1.4 mM MgAcetate, 0.5 mM DTT, 100 ug/ml BSA, 0.1%Tween-20, 0.2 U/ul Klenow exo-polymerase, and 0.5 uM dNTPs with a 1:1ratio of biotin-11 labeled nucleotides to “cold” nucleotides for dCTP,dGTP, and dUTP. In the experiments with SSB the concentration was 0.2ug/20 ul rxn. Array-based Klentaq conditions are described in ExampleIII.

FIG. 14A shows a scatter plot for an ASPE, reactions run with Klenowpolymerase BeadArrays™ in the presence of SSB and absence of a targetnucleic acid sample (ntc=no target control). As demonstrated by FIG.14C, ectopic signal was greatly reduced in the presence of SSB comparedto in the absence of SSB. Similar results were obtained for ASPEreactions run with Klentaq polymerase. The plots shown in FIGS. 14C andD were obtained by sorting signals from scatter plots along the X-axisaccording to increasing intensity. As shown in FIG. 14B, allele specificextension occurred at detectable levels for ASPE reactions carried outin the presence of a target sample containing an amplified population ofgenome fragments.

These results demonstrate that the inclusion of SSB in a primerextension assay suppresses ectopic extension while maintaining orimproving allele-specific extension. Further studies have indicated thatinclusion of SSB in an array-based ASPE reaction improved the allelicdiscrimination.

EXAMPLE VII Evaluation of Genome Fragment Populations Produced by RandomPrimer Amplification

This example demonstrates that human genome fragment populationsproduced by random primer amplification (RPA) are representative oftheir genome templates, having little allelic bias and are capable ofbeing reproducibly generated.

RPA reactions were used to produce amplified populations of genomefragments from human genomic DNA using methods described in Example V.The amplification reactions were carried out in a single tube formatwithout the need for isolation of reaction components or products priorto incubating the reaction mixtures with probe arrays. With theexception of modifications described below, the reaction mixtures wereincubated with BeadArrays™ as described in Example V and detection wascarried out using ASPE as described in Example

The results shown in FIG. 15 illustrate the representation achieved inthe amplification process. Duplicate RPA reactions carried out on 100 ngof human genomic DNA (Corlett Cell Repositories, Camden, N.J.) in 100 ulyielded populations of genome fragments having 1-2 ug DNA/ul. Duplicateunamplified genome samples consisted of human placental DNA(Sigma-Aldrich, Part No. D3287) that was fragmented with DNAse Ito anaverage size of about 200 to 300 bases.

Amplified and unamplified samples were hybridized to arrays with probesdesigned to non-polymorphic regions of the genome. As such, all probeswere perfect matches to the genome and should extend in the genotypingassay. The intensity values obtained for individual probes followinghybridization to two different samples are plotted in the scatter plotsof FIG. 15 . As shown in FIG. 15A., a high degree of correlationoccurred between duplicate unamplified samples. Similarly and as shownin FIG. 15B, strong correlation was observed between duplicate amplifiedsamples, indicating that the amplification methods gave highlyreproducible results. The amplified vs. unamplified scatterplot of FIG.15C, showed a more diffuse cluster compared to those observed for theduplicates and indicates that some loci were over-represented whereasothers were under represented in the amplified sample.

Nevertheless, the results indicated good representation. The number ofprobes (counts) having particular ratios of signal intensities forunamplified to amplified DNA inputs (ratio of amplified:unamplified) isplotted in FIG. 16A. The data demonstrated that 90.1% of the detectedloci had an intensity variance in the amplified population that did notexceed 0.5- to 2-fold compared to the intensity measured for unamplifiedgenomic DNA. Thus, 90.1% of the detected loci in the amplifiedpopulation were represented in no less than 0.5 fold shortage and nomore than 2 fold excess compared to their relative amounts in theunamplified genome. Furthermore, 97.4% of detected loci in the amplifiedpopulation were represented in no less than 0.3 fold shortage and nomore than 3-fold excess compared to their relative amounts in theunamplified genome.

The representationally amplified population of genome fragments wascompared to unamplified control DNA samples in the GoldenGate™ assay(Illumina, Inc. San Diego, Calif.). Exemplary data for four loci (1824,2706, 3633 and 6126) is shown in the Genoplots (also called GenTrainplots) of FIG. 17 . The genoplots are polar coordinate replots ofstandard genotyping scatter plots. Standard genotyping scatter plotshave an axis of intensity detected for a first channel (correlated witha first allele) vs. intensity detected for a second color channel(correlated with a second allele) and plot a scatterpoint for each locusaccording to its intensity in each channel. Genoplots are replots ofeach scatter point according to the distance of a line drawn from theorigin to the scatter point (R) and the angle between the line and the xaxis (theta). As shown in FIG. 17 , scatterpoints for data generatedfrom RPA mixtures produced from 10 ng, 100 ng or 1 ug genome inputsresulted in good clusters compared to control clusters (circled) fromunamplified genomic DNA, indicating very little allelic bias.

The limit of detection (LOD) in genotyping assays was shown to increaseas increasing amounts of genomic DNA were input into RPA reactions.Separate RPA reactions were carried out (in duplicate) with variousamounts of input genomic DNA. The input amounts were in the range of 1femtogram to 100 nanograms, including the amounts plotted on the x-axisof FIG. 18A. FIG. 18A is a bar graph showing the average intensitydetected for all probes on each array (LOD) following hybridization andASPE detection of RPA reaction mixtures generated from different amountsof input genomic DNA (input). As shown in FIG. 18A amounts of inputgenomic DNA of 10 pg (approximately 3 copies of the human genome) orgreater resulted in LOD values that were substantially increasedcompared to a control RPA reaction in which no input genomic DNA wasused (0 g). LOD was substantially increased over background when atleast 100 pg (30 genome copies), 1 ng (300 genome copies), 10 ng (3,000genome copies) or 100 ng (30,000 genome copies) of input human genomicDNA was used for the RPA reaction as shown in FIG. 18A.

Representation was shown to improve as increasing amounts of genomic DNAwere input into RPA reactions. The bar graph shown in FIG. 18B plotsPM/(PM+MIN4) for all probes of an array (ratio) when used to probe RPAmixtures produced from varying amounts of input genomic DNA (input).Amounts of input genomic DNA of 10 pg (approximately 3 copies of thehuman gnome) or greater resulted in a substantial improvement inrepresentation when compared to a control RPA reaction in which no inputgenomic DNA (0 g) or low levels of genomic DNA (femtogram amounts) wereused. Representation was further substantially improved when at least100 pg (30 genome copies), 1 ng (300 genome copies), 10 ng (3,000 genomecopies) or 100 ng (30,000 genome copies) of input human genomic DNA wasused for the RPA reaction.

These results indicate that RPA can be used to produce hundreds ofmicrograms of an amplified population of genome fragments fromquantities of genomic DNA template as low as a few picograms. Theamplified populations of genome fragments produced by RPA have goodrepresentation, can be reproducibly made and have little allelic bias.Thus, the DNA produced by RPA is of sufficient quantity and quality forwhole genome genotyping.

EXAMPLE VIII Whole Genome Genotyping Assay Performance

This Example demonstrates that whole genome genotyping of an amplifiedpopulation of genome fragments by direct hybridization to a DNA arrayand array-based primer extension produces accurate, high quality SNPscoring results for human subjects.

Genomic DNA (100 ng) was obtained from 95 samples in the Centre d'Etudedu Polymorphisme Humain (CEPH) in the set used for quality control ofthe International HapMap project (for sample information seeinternational HapMap Consortium, Nature 426:789-796 (2003)). RPAreactions were carried out as described in Example V, resulting inreaction mixtures, containing 188 ug of DNA in 100 ul. The undilutedreaction mixtures were incubated with the BeadArrays™ having 50-merprobes specific for the 1500 HapMap QC set of loci (for loci informationsee International HapMap Consortium, Nature 426:789-796 (2003)) usingmethods described in Example V followed by ASPE as described in ExampleIII. Arrays were then imaged on a charge coupled device reader asdescribed in Gunderson et al., Genome Res. 14:870-877 (2004). SNPgenotypes were called using GenCall software (Illumina Inc., San Diego,Calif.).

FIGS. 19A and 19B show representative Genoplots (also called GenTrainplots the 860 and 954 loci, respectively. Good duster separation wasObtained for the 860 and 954 loci, yielding gene cluster scores (GCS) of7.5 and 4.4, respectively (GCS=Min[(Abs(θ_(AB)−θ_(AA))/(σ_(AB)+∝_(AA))),(Abs(θ_(AB)−θ_(BB))/(σ_(AB)+σ_(BB))], where θ_(AB) is the average θ forthe AB cluster (6 is described above in regard to FIG. 11 ) and σ_(AB)is the standard deviation of θ_(AB)). FIG. 19C shows a distribution ofloci according to genotype cluster separation score. Over 75% of locihad a GCS of 3.0 or higher (dark bars) and were, therefore, consideredacceptable for genotyping.

A summary of genotyping statistics for interrogation of HapMap QC set ofloci in the CEPH samples is shown in Table 1. Assay conversion rate wasassessed by counting the number of loci that successfully detected aminor allele. Non-polymorphic loci and high-copy number loci werecounted as assay failures in regard to developing a real SNP assay.Technically, many of the non-polymorphic loci were successful assays,but they were not counted because they did not exhibit a minor allele.The assay conversion rate compared to results from the Golden Gate Assay(Illumina, Inc. San Diego, Calif.) using the same genomic DNA sampleswas 95%. The call rate was quite high at 99.5% and the reproducibilitywas greater than 99.99%.

Concordance was determined between the genotyping results obtained asdescribed above and genotyping results obtained for the same samples andloci using the Golden Gate Assay (Illumina, Inc. San Diego, Calif.).Concordance was greater than 99.9%.

Parameter Values Percent Assay Conversion 819/864  95% Call Rate68807/68970 99.5% Reproducibility 8189/8190 99.99%  Concordance137,456/137,614 99.9%

These results indicate that the whole genome genotyping assay provideshigh quality genotyping data, on par with the Golden Gate assay which iscurrently being used for genotyping a large portion of the genome in theinternational HapMap project.

EXAMPLE IX Stripping Arrays to Remove Hybridized Target Prior toDetection

This example demonstrates removal of hybridized target from an array bystripping with 0.1N NaOH after modification of probes bytarget-dependent polymerase extension.

Genomic DNA was obtained from Corlett Cell Repositories (Camden, N.J.).RPA reactions were carried out as described in Example VII. Theresulting reaction mixtures were hybridized to BeadArrays™ and ASPEreactions performed as described in Example III. Following the ASPEreaction and prior to detection of fluorescent signal the arrays weretreated with 0.1 N NaOH in water (+NaOH) or 1× hybridization buffer,lacking formamide (—NaOH). Arrays were detected as described in ExampleVIII.

As shown in FIG. 20 , post-extension stripping of the array with NaOHreduced background signal from the mismatch probes, and resulted in alarger ratiometric difference between signal from mismatch and perfectmatch probes.

These results indicate that stripping arrays after probe modificationalthough not necessary can be used to greatly improve assay specificity.

EXAMPLE X Whole Genome Amplification of Bisulfite Treated DNA

This example describes methods to whole genome amplify bisulfite-treatedDNA. Typically bisulfite treatment of DNA generates substantialdepurination and concomitant fragmentation of the DNA. This fragmentedproduct is typically amplified in low yield using strand-displacingpolymerases in random primer whole genome amplification approaches. Twoapproaches for improving amplification yield are described here. Thefirst approach is concatenation of the fragmented sample and use of thelonger concatenated products as templates for strand-displacement randomprimer amplification. The second approach creates a representation outof the fragmented targets by attachment of universal priming sites tothe ends of the fragments.

Bisulfite treatment of genomic DNA is typically used for detectingmethylation based on a reaction in which cytosine is converted touracil, but 5-methylcytosine remains non-reactive (see, for example,Feil et al. Nucleic Acids Res, 22; 695-696 (1994); Frommer et al., ProcNatl Acad Sci USA, 89; 1827-1831 (1992)). A further reaction of DNA withbisulfite is depurination and concomitant fragmentation. The DNAfragments produced by bisulfite treatment contain a phosphate group atthe 3′ terminus. This phosphate group effectively blocks reaction of the3′ terminus with single nucleotides or polynucleotides using severalbiological enzymes.

Concatenation of Bisulfite Treated Genomic DNA

The 3′ phosphate group of bisulfite treated genomic DNA is removed bytreatment with alkaline phosphatase or the 3′ phosphatase activity of T4DNA kinase using standard conditions recommended by the supplier. T4 DNAkinase maintains the 5′ phosphate intact while removing the 3′ phosphate(in the presence of ATP), resulting in a product having a 5′ phosphateand 3′ hydroxyl (see FIG. 21A) In contrast, alkaline phosphatase removesboth the 5′ and 3′ phosphate, resulting in a product having both 3′ and5′ hydroxyls (see FIG. 21A).

After removal of the 3′ phosphate by T4 DNA kinase, the products arethen incubated with T4 RNA ligase to create concatamers using conditionsdescribed in McCoy et al., supra (1980). The resulting linear andcircular concatamers having various sizes are amplified by random primeramplification as described herein, for example, in Example V. Thisamplified product is then used for genotyping as described herein, forexample, in Example VII, and provides a means for conducting genome widemethylation profiling.

Tailing of Bisulfite Treated Genomic DNA

The 3′ phosphates of bisulfite treated fragments are converted into 3′hydroxyls as described above. Universal tails are added to the productusing one of three different methods.

The first method is treatment of DNA fragments with terminaldeoxynucleotide transferase (TdT) and dGTP to add a polyguanylate tailto the 3′ end (see FIG. 21C). A universal tail is added to the 5′ end ofthe fragment incubation with DNA ligase and an oligonucleotide having a3′ random 4-mer duplex adapter and a 5′ universal priming site sequence(FIG. 21C) using standard conditions recommended by the supplier. Theresulting fragments are amplified by polymerase chain reaction using auniversal primer (primer A in FIG. 21C) that complements the 5′universal priming site tail of the fragments and a polycytidylate primer(primer B in FIG. 21C) that complements the 3′ polyguanylate tail of thefragments.

In the second method a 5′ tail is added by T4 RNA ligase-mediatedligation of an oligonucleotide having a universal priming site usingstandard conditions recommended by the supplier. As shown in FIG. 21D,the reaction is carried out in two steps. In the first step, a universalpriming site oligonucleotide having a 5′ phosphate but lacking a 3′hydroxyl is reacted with the fragment such that a 3′ tail is added tothe fragment. In the second step, a universal priming siteoligonucleotide having a 3′ hydroxyl but lacking a 5′ phosphate isreacted with the fragment such that a 5′ tail is added to the fragment.The use of blocked oligonucleotides in two steps reduces unwanted sidereactions due to self-ligation of the universal priming siteoligonucleotides. The resulting fragments are amplified by polymerasechain reaction using a universal primer (primer A in FIG. 21D) thatcomplements the 5′ universal priming site tail of the fragments and auniversal primer (primer B in FIG. 21D) that complements the 3′universal priming site of the fragments. This amplified product is thenused for genotyping as described herein, for example, in Example VII,and provides a means for conducting genome wide methylation profiling.

The third method employs direct ligation of oligonucleotides havinguniversal priming sites to both the 3′ and 5′ termini using T4 RNApolymerase using standard conditions recommended by the supplier.Complementary universal primers are then used to amplify the fragmentsby polymerase chain reaction. This amplified product is then used forgenotyping as described herein, for example, in Example VII, andprovides a means for conducting genome wide methylation profiling.

Throughout this application various publications, patents and patentapplications have been referenced. The disclosure of these publicationspatents and patent applications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains.

The term “comprising” is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements,

Various embodiments of the invention have been described broadly andgenerically herein. Each of the narrower species and subgenericgroupings falling within the generic disclosure also form the part ofthese inventions. This includes within the generic description of eachof the inventions a proviso or negative limitation that will allowremoving any subject matter from the genus, regardless or whether or notthe material to be removed was specifically recited.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the invention. Accordingly, the inventionis limited only by the claims.

What is claimed is:
 1. A method for detecting typable loci of a genome,comprising the steps of (a) in vitro transcribing a population ofamplified genome fragments, thereby obtaining genomic RNA fragments; (b)hybridizing said genomic RNA fragments with a plurality of nucleic acidprobes having sequences corresponding to said typable loci, therebyforming a plurality of RNA fragment-probe hybrids; (c) replicating saidgenomic RNA fragment-probe hybrids with a plurality of differentlocus-specific primers, thereby producing a locus-specific, amplifiedrepresentative population of genome fragments and (d) detecting typableloci of said RNA fragment-probe hybrids.
 2. The method of claim 1,wherein said population of amplified genome fragments is produced byamplification with a plurality of random primers.
 3. The method of claim1, wherein step (a) comprises in vitro transcribing said population ofamplified genome fragments using random primers comprising a 3′ sequenceregion that is random and another sequence region having a constantsequence, thereby obtaining genomic RNA fragments labeled with saidconstant sequence.
 4. The method of claim 3, wherein said locus-specificprimers comprise a 3′ sequence region that is locus-specific and anothersequence region having a second constant sequence, thereby obtaininggenomic RNA fragments labeled with said first constant region and saidsecond constant region.
 5. The method of claim 4, further comprising astep of replicating the genomic RNA fragments with complementary primersto the first constant region and second constant region.
 6. The methodof claim 1, wherein replicating said genomic RNA fragment-probe hybridsoccurs under conditions wherein DNA-dependent DNA synthesis isinhibited.
 7. The method of claim 1, further comprising a step ofisolating said genomic RNA fragments.
 8. A method of producing a reducedcomplexity, locus-specific, amplified representative population ofgenome fragments, comprising the steps of (a) replicating a nativegenome with a plurality of random primers, thereby producing anamplified representative population of genome fragments; (b) hybridizingthe amplified representative population of genome fragments with asubstrate comprising a plurality of different locus-specific primerscomprising 3′ sequence and a 5′ universal tail; (c) performing primerextension to replicate a sub-population of said amplified representativepopulation of genome fragments with the plurality of differentlocus-specific primers, thereby producing a locus-specific, amplifiedrepresentative population of genome fragments; and (d) isolating saidsub-population, thereby producing a reduced complexity, locus-specific,amplified representative population of genome fragments.
 9. The methodof claim 8, wherein said random primers comprise a 3′ sequence regionthat is random and a 5′ sequence region having a first constantsequence, thereby producing a reduced complexity, locus-specific,amplified representative population of genome fragments labeled withsaid constant sequence.
 10. The method of claim 9, wherein saidlocus-specific primers comprise a 3′ sequence region that islocus-specific and a 5′ sequence region having a second constantsequence, thereby producing a locus-specific, amplified representativepopulation of genome fragments labeled with said first constant regionand said second constant region.
 11. The method of claim 10, furthercomprising a step of replicating the reduced complexity, locus specific,amplified representative population of genome fragments withcomplementary primers to said first constant region and said secondconstant region.
 12. The method of claim 8, further comprising a step ofisolating said amplified representative population of genome fragments.